Survivin, a protein that inhibits cellular apoptosis, and its modulation

ABSTRACT

The present invention provides the amino acid of a protein that inhibits cellular apoptosis, herein termed the Survivin protein and nucleic acid molecules that encode Survivin. Based on this disclosure, the present invention provides isolated Survivin protein, isolated Survivin encoding nucleic acid molecules, methods of isolating other members of the Survivin family of proteins, methods for identifying agents that block Survivin mediated inhibition of cellular apoptosis, methods of using agents that block Survivin mediated inhibition or Survivin expression to modulate biological and pathological processes, and methods of assaying Survivin activity.

STATEMENT OF RELATED APPLICATIONS

[0001] This application is based upon provisional U.S. ApplicationSerial No. 60/031,435, filed Nov. 20, 1996, the disclosure of which ishereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of modulating cellapoptosis, particularly agents useful to inhibit apoptosis, as well asto diagnostic and prognostic assays involving conditions in mediated bythe expression of inhibitors of apoptosis. The invention specificallyrelates to the identification of a novel human gene, tentatively namedSurvivin. Survivin encodes a protein, Survivin, that inhibits cellularapoptosis, particularly in cancer cells and embryonic cells.

BACKGROUND OF THE INVENTION

[0003] Regulation of cell proliferation by programmed cell death(apoptosis) maintains tissue homeostasis during development anddifferentiation (Raff, M. D., Nature (1992) 356:397400; Vaux, D. L. etal., Cell (1994) 76:777-779). This process involves an evolutionarilyconserved multi-step cascade (Oltvai, Z. et al., Cell (1994)79:189-192), and is controlled by proteins that promote or counteractapoptotic cell death Apoptosis also involves cell surface receptors(Smith, A. et al., Cell (1994) 76, 959-962), and associated signaltransducers (Tartaglia, L. A. et al., Immunol Today (1992) 13:151-153),protease gene families (Martin, S. J. et al., Cell (1995) 82:349-352),intracellular second messengers (Kroemer, G. et al., FASEB J (1995)9:1277-1287), tumor suppressor genes (Haffner, R. et al., Curr Op GenDev (1995) 5:84-90), and negative regulatory proteins that counteractapoptotic cell death (Hockenbery, D. et al., Nature (1990) 348:334-336).Aberrantly increased apoptosis or abnormally prolonged cell survival(Oltvai, Z. N. et al., Cell (1994) 79:189-192) may both contribute tothe pathogenesis of human diseases, including autoimmune disorders,neurodegenerative processes, and cancer (Steller, H., Science (1995)267:1445-1449; Thompson, C. B., Science (1995) 267:1456-1462).

[0004] Specifically, for example, inhibitors of apoptosis, most notablyof the bcl-2 family (Reed, J, J Cell Biol (1994) 124:1-6, and Yang, E,et al., Blood (1996) 88:386-401), maintain lymphoid homeostasis andmorphogenesis in adult (Hockenbery, D et al., Proc Natl Acad Sci USA(1991) 88:6961-6965) and fetal (LeBrun, D. et al. (1993) 142:743-753)tissues. Deregulated expression of bcl-2 has also been implicated incancer, by aberrantly prolonging cell survival and facilitating theinsurgence of transforming mutations.

[0005] In addition to bcl-2, several members of a new gene family ofinhibitors of apoptosis related to the baculovirus IAP gene (Bimbaum, M.J. et al., J Virology (1994) 68:2521-2528; Clem, R. J. et al., Mol CellBiol (1994) 14:5212-5222) have been identified in Drosophila andmammalian cells (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay,B. A. et al., Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996)379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al.,Cell (1995) 80:167-178). These molecules are highly conservedevolutionarily; they share a similar architecture organized in two orthree approximately 70 amino acid amino terminus Cys/His baculovirus IAPrepeats (BIR) and by a carboxy terminus zinc-binding domain, designatedRING finger (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B.A. et al., Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996)379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al.,Cell (1995) 80:167-178). Recombinant expression of IAP proteins blocksapoptosis induced by various stimuli in vitro (Duckett, C. S. et al.,EMBO J (1996) 15:2685-2694; Liston, P. et al., Nature (1996)379:349-353), and promotes abnormally prolonged cell survival in thedevelopmentally-regulated model of the Drosophila eye, in vivo (Hay, B.A. et al., Cell (1995) 83:1253-1262). Finally, deletions in a IAPneuronal inhibitor of apoptosis, NAIP, were reported in 75% of patientswith spinal muscular atrophy, thus suggesting a potential role of thisgene family in human diseases (Roy, N. et al., Cell (1995) 80:167-178).

[0006] Therapeutic and diagnostic uses of nucleic acids that encodevarious inhibitors of apoptosis relating to a member of the IAP familyhave been described in the patent literature. See, for example,International Patent Applications No. WO 97/06255, WO 97/26331, and WO97/32601. In particular, the uses of such genes and gene products arecontemplated for the novel protein and its encoding nucleic aciddiscusssed below.

[0007] Recently, a novel gene encoding a structurally unique IAPapoptosis inhibitor, designated Survivin has been identified. Survivinis a −16.5 kD cytoplasmic protein containing a single BIR, and a highlycharged carboxyl-terminus coiled-coil region instead of a RING finger,which inhibits apoptosis induced by growth factor (IL-3) withdrawal whentransferred in B cell precursors (Ambrosini, G. et al., Nature Med.(1997) 3:917-921). At variance with bcl-2 or other IAP proteins,Survivin is undetectable in adult tissues, but becomes prominentlyexpressed in all the most common human cancers of lung, colon, breast,pancreas, and prostate, and in −50% of high-grade non-Hodgkin'slymphomas, in vivo. Intriguingly, the coding strand of the Survivin genewas highly homologous to the sequence of Effector cell ProteaseReceptor-1 (EPR-1) (Altieri, D. C., FASEB J (1995) 9:860-865), butoriented in the opposite direction, thus suggesting the existence of twoseparate genes duplicated in a head-to-head configuration.

[0008] The present invention is based on the identification of a novelhuman gene which is nearly identical to EPR-1, but oriented in theopposite direction. The antisense EPR-1 gene product, designatedSurvivin, is a distantly related member of the IAP family of inhibitorsof apoptosis (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B.A. et al., Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996)379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al.,Cell (1995) 80:167-178), and is prominently expressed in activelyproliferating transformed cells and in common human cancers, in vivo,but not in adjacent normal cells. Functionally, inhibition of Survivinexpression by up-regulating its natural antisense EPR-1 transcriptresulted in massive apoptosis and decreased cell growth.

SUMMARY OF THE INVENTION

[0009] The present invention is based, in part, on the isolation andidentification of a protein that is expressed in most cancer cells andinhibits cellular apoptosis, hereinafter Survivin or the Survivinprotein. Based on this observation, the present invention providespurified Survivin protein.

[0010] The present invention further provides nucleic acid moleculesthat encode the Survivin protein. Such nucleic acid molecules can be inan isolated form, or can be operably linked to expression controlelements or vector sequences.

[0011] The present invention further provides methods of identifyingother members of the Survivin family of proteins. Specifically, thenucleic acid sequence of Survivin can be used as a probe, or to generatePCR primers, in methods to identify nucleic acid molecules that encodeother members of the Survivin family of proteins.

[0012] The present invention further provides antibodies that bind toSurvivin. Such antibodies can be either polyclonal or monoclonal.Anti-Survivin antibodies can be used in a variety of diagnostic formatsand for a variety of therapeutic methods.

[0013] The present invention further provides methods for isolatingSurvivin binding partners. Survivin binding partners are isolated usingthe Survivin protein as a capture probe. Alternatively, Survivin can beused as bait in the yeast two-hybrid system to screen an expressionlibrary and identify genes that encode proteins that bind to theSurvivin protein Binding partners isolated by these methods are usefulin preparing antibodies and also serve as targets for drug development.

[0014] The present invention further provides methods to identify agentsthat can block or modulate the association of Survivin with a bindingpartner. Specifically, an agent can be tested for the ability to block,reduce or otherwise modulate the association of Survivin with a bindingpartner by contacting Survivin, or a fragment thereof, and a bindingpartner with a test agent and determining whether the test agent blocksor reduces the binding of the Survivin protein to the binding partner.

[0015] The present invention further provides methods for reducing orblocking the association of Survivin with one or more of its bindingpartners. Specifically, the association of Survivin with a bindingpartner can be blocked or reduced by contacting Survivin, or the bindingpartner, with an agent that blocks the binding of Survivin to thebinding partner. The method can utilize an agent that binds to Survivinor to the binding partner.

[0016] The present invention further provides methods of regulating theexpression of Survivin within a cell. Expression of Survivin within acell can be regulated so as to produce or inhibit the production ofSurvivin.

[0017] Blocking Survivin/binding partner associations or Survivinexpression can be used to modulate biological and pathological processesthat require Survivin. For example, methods that reduce Survivinproduction induce apoptosis of tumor cells. Stimulation of Survivinproduction can be used as a means of extending the culturability ofcells or tissues.

[0018] The biological and pathological processes that require Survivinor Survivin/binding partner interactions can further be modulated usinggene therapy methods. Additional genetic manipulation within an organismcan be used to alter the expression of a Survivin gene or the productionof a Survivin protein in an animal model. For example, a Survivin genecan be altered to correct a genetic deficiency; peptide modulators ofSurvivin activity can be produced within a target cell using genetictransformation methods to introduce a modulator encoding nucleic acidmolecules into a target cell; etc. The use of nucleic acids forantisense and triple helix therapies and interventions are expresslycontemplated.

[0019] The present invention further provides methods of reducing theseverity of pathological processes that require Survivin. Sinceexpression of Survivin or association of Survivin with a binding partneris required for Survivin-mediated biological processes, agents thatblock Survivin expression, Survivin activity or the association ofSurvivin with a binding partner, can be used in therapeutic methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 shows the identification of a complementary EPR-1 gene. A,B. Chromosomal location. A digoxigenin-labeled human P1 genomic cloneselected by hybridization with the EPR-1 cDNA, was incubated withmetaphase chromosomes isolated from phytohemagglutinin-stimulated PBMCin 50% formamide, 10% dextran sulfate and 2×SSC. The EPR-1-hybridizinggene was mapped in single-color labeling to the long arm of a group Echromosome (A, green staining), and in two-color staining with probeD17Z1, specific for the centromere of chromosome 17 (B, red staining),to the long arm of chromosome 17 (B, green staining), to band 17q25. C.Map of the antisense EPR-1 gene. A contig spanning 14796 bp was derivedfrom two EPR-1-hybridizing P1 clones, subcloned in pBSKS⁻, andcompletely sequenced on both strands. Orientation of the map is 5′→3′with respect to the position of intron-exon boundaries (see below).Exons are solid boxes, a putative CpG island upstream exon 1 is an openbox. The translational initiation codon (ATG) is indicated. Restrictionsites are: B, BamHI, H, HindIII; P, PstI; S, SmaI; X, XbaI. D.Intron-exon boundaries of the antisense EPR-1 gene. Positions of theintron-exon boundaries in bp are indicated in parenthesis.

[0021]FIG. 2 shows the complexity and evolutionary conservation ofEPR-1-related sequences. A. Southern blot of human genomic DNA. Sampleswere digested with the indicated restriction enzymes, transferred toGeneScreen nylon membranes and hybridized with the EPR-1 cDNA, in 5×SSC,0.5% SDS, 5×Denhardt's and 0.1% sodium pyrophosphate at 65° C.Radioactive bands indicated by an arrow (7.6 kb BamHI, 7.5 kb XbaI andHindIII fragments of 15, 7.5, 6.4, and 3.7 kb) do not derive from theantisense EPR-1 gene in FIG. 1C. B. Southern blot of pulsed field gelelectrophoresis. High molecular weight human genomic DNA was digestedwith the indicated restriction enzymes, separated by pulsed field gelelectrophoresis for 20 h at 200 V with a pulse time of 75 sec,transferred to nylon membrane, and hybridized with the EPR-1 cDNA, asdescribed in A. C. Multiple species Southern blot. EcoRI-digestedgenomic DNA from the indicated species was hybridized with a 3′ 548 bpfragment of the EPR-1 cDNA, as described in A. For all panels, molecularweight markers in kb are shown on the left.

[0022]FIG. 3 shows the discordant tissue distribution of sense/antisenseEPR-1 transcipts. Northern hybridization was carried out on a multipletissue adult or fetal mRNA blot with single strand-specific probes in5×SSPE, 10×Denhardt's solution, 2% SDS, 100 mg/ml denatured salmon spermDNA at 60° C. for 14 h. After washes in 2×SSC at 60° C. and in 0.2×SSCat 22° C., radioactive bands were visualized by autoradiography. A.EPR-1-specific single-strand probe. B. Antisense EPR-1-specificsingle-strand probe. C. Control actin probe. Molecular weight markers inkb are shown on the left.

[0023]FIG. 4 shows the sequence analysis of Survivin and expression incell lines. A. Predicted translation of the antisense EPR-1 gene product(Survivin). B. Sequence alignment of the BIR in Survivin and in otherIAP proteins by the Clustal method. IAP proteins are identified byaccession number, L49433, TNFR2-TRAF signaling complex-associated IAP;L49441, apoptosis 2 inhibitor (Drosophila); P41436, IAP gene from Cydiapomonella granulosis virus; P41437, IAP gene from Orgya pseudotsugatanuclear polyhedrosis virus; U19251, NAIP, neuronal inhibitor ofapoptosis; U32373, IAP-like protein ILP from Drosophila melanogaster;U32974, human IAP-like protein ILP; U36842, mouse inhibitor ofapoptosis; U45878, human inhibitor of apoptosis 1; U45879, humaninhibitor of apoptosis 2; U45880, X-linked inhibitor of apoptosis;U45881, Drosophila inhibitor of apoptosis. Conserved residues are boxed,identities between Survivin and NAIP (U19251) are boxed and shaded. C.Immunoblotting with anti-Survivin antibody JC700. Protein-normalizedaliquots of SDS-extracts of cell lines HEL (erythroleukemia), Daudi andJY (B lymphoma), THP-1 (monocytic), Jurkat and MOLT13 (T leukemia), ornon transformed human lung Lu18 fibroblasts, HUVEC or PBMC wereseparated by electrophoresis on a 5-20% SDS gradient gel, transferred toImmobilon and immunoblotted with control non-immune rabbit IgG (RbIgG),or anti-Survivin antibody JC700 (Survivin). Protein bands werevisualized by alkaline phosphatase-conjugated goat anti-rabbit IgG andtetrazolium salts. Molecular weight markers in kDa are shown on theleft.

[0024]FIG. 5 shows the regulation of Survivin expression by cellgrowth/differentiation. HL-60 cells were terminally differentiated to amature monocytic phenotype by a 72 h culture with 0.1 mM vitamin D₃ plus17.8 mg/ml indomethacin. Survivin expression before or after vitamin D₃differentiation was detected by immunoblotting with JC700 antibody, orby Northern hybridization with a Survivin-specific single strand probe.RbIgG, control non-immune rabbit IgG. Protein molecular weight markersin kDa and position of ribosomal bands are shown on the left of eachblot.

[0025]FIG. 6 shows the over-expression of Survivin in human cancer, invivo. A. Immunohistochemical staining of human lung adenocarcinoma withaffinity-purified anti-Survivin antibody JC700 (20 μg/ml). B. Inhibitionof JC700 staining of lung adenocarcinoma by pre-absorption with theimmunizing Survivin 3-19 peptide. C. Immunohistochemical expression ofSurvivin in squamous lung cell carcinoma, but not in the adjacent normalgland epithelium of the lung (C, arrow). D. In-situ hybridization ofSurvivin mRNA in squamous lung cell carcinoma with a Survivin-specificriboprobe. E. Expression of Survivin in pancreatic adenocarcinoma byimmunohistochemistry with JC700. F. Normal pancreas, negative forSurvivin expression by immunohistochemistry. G. In situ hybridization ofSurvivin mRNA expression in colon adenocarcinoma, but H, not in theadjacent non neoplastic colon gland epithelium (H, arrow).Magnifications are ×200, except G, ×400.

[0026]FIG. 7 shows the effect of Survivin on apoptosis/proliferation. A.EPR-1-regulation of Survivin expression. HeLa cells were transfectedwith control vector pML1 or the EPR-1 cDNA (which is antisense toSurvivin) by electroporation, and selected in hygromicin (0.4 mg/ml).Aliquots of vector control HeLa cells (Vector) or Survivin antisensetransfectants (Antisense) were induced with 200 mM ZnSO₄detergent-solubilized, and immunoblotted with the anti-Survivin JC700antibody. Molecular weight markers are shown on the left. B. Effect ofSurvivin on apoptosis. Survivin antisense transfectants (1, 2), orvector control HeLa cells (3, 4) were induced with Zn²⁺ ions in 0% FBSfor 24 h and stained by the AptoTag method with TdT-catalyzed dUTPlabeling of 3′-OH DNA ends and immunoperoxidase (1, 3), or byhematoxylin-eosin (HE) (2, 4). 1. Prominent nuclear DNA fragmentationdetected by AptoTag staining in serum-starved Survivin antisensetransfectants; 2. HE staining of antisense transfectants reveals thepresence of numerous apoptotic bodies (arrows); 3. AptoTag staining ofvector control HeLa cells detects a few sparse apoptotic cell (arrow);4. HE staining of vector control HeLa cells. The arrow indicates asingle apoptotic body. Magnification ×400. C. Effect of Survivin on cellgrowth. Twenty thousands vector control HeLa cells (Vector) or Survivinantisense transfectants (Antisense) were seeded in 24-well plates,induced with ZnSO₄, harvested at the indicated time points, and cellproliferation was determined microscopically by direct cell count. Dataare the mean±SEM of replicates of a representative experiment out ofseven independent determinations.

[0027]FIG. 8 shows the expression of Survivin in HL-60 cells. HL-60cells were examined via Western and Northern blots for Survivinexpression.

[0028]FIG. 9 presents a structural analysis of Survivin. The Survivinprotein was analyzed using the Chou-Fasman, Garnier-Robson,Kyle-Doolittle, Eisenberg, Karplus-Schultz, Jameson-Wolf and Eminianalysis methods.

[0029]FIG. 10 shows the nucleotide Sequence of Survivin.

[0030]FIG. 11 shows the expression of Survivin and the generation andcharacterization of anti-Survivin mAb 8E2 by ELISA and immunoblotting.

[0031]FIG. 12 shows the site-directed mutagenesis of Survivin andidentification of key functional residues involved in apoptosisinhibition.

[0032]FIG. 13 shows the cytoprotective effect of Survivin addition onendothelial cell apoptosis.

[0033]FIG. 14 shows that the presence of Survivin is a negativepredictive-prognostic factor in neuroblastoma

[0034]FIG. 15 shows that the presence of Survivin is a negativepredictive prognostic factor in high-grade non-Hodgkin's lymphoma.

[0035]FIG. 16 shows the down regulation of Survivin induced byinflammatory and cytostatic cytokines.

[0036]FIG. 17 shows the effects of Survivin constructs or XIAP onapoptosis induced in NIH3T3 cells by hydrogen peroxide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0037] I. General Description

[0038] The present invention is based in part on identifying a novelprotein that is expressed in tumor cells and inhibits cellularapoptosis, hereinafter the Survivin protein or Survivin. Survivin isalso found to be expressed in embryonic tissues.

[0039] The Survivin protein can be used as an agent, or serve as atarget for agents, that can be used to inhibit or stimulate Survivinmediated inhibition of cellular apoptosis, for example to block abnormalcell growth or to extend cell growth in culture.

[0040] As used herein, modulation of apoptosis means increasing ordecreasing the number of cells that would otherwise undergo apoptosis ina given cell population. This can be effected by increasing ordecreasing the amount of Survivin present in a cell or by increasing ordecreasing the activity of the Survivin. Preferably, the given cellpopulation in which apoptosis is to be modulated is found in a tumor orother tissue or group of cells in which beneficial effect results fromthe modulation. Also, preferably, the increase or decrease in number ofcells that would otherwise undergo apoptosis in a given cell populationis at least about 10%, 20%, 40% or more preferably at least about 50% ofthe cells in that population.

[0041] The present invention is further based on the development ofmethods for isolating proteins that bind to Survivin. Probes based onthe Survivin protein or fragments of Survivin as discussed below areused as capture probes to isolate Survivin binding proteins. Dominantnegative proteins, DNAs encoding these proteins, antibodies to thesebinding proteins, peptide fragments of these proteins or mimics of theseproteins may be introduced into cells to affect Survivin function.Additionally, these proteins provide novel targets for screening ofsynthetic small molecules and combinatorial or naturally occurringcompound libraries to discover novel therapeutics to regulate Survivinfunction.

[0042] II. Identification, General Characterization and TissueDistribution of Survivin

[0043] The present invention is based on the identification onchromosome 17q25 of a novel member of the IAP family of inhibitors ofapoptosis, designated Survivin, which may confer a selective advantagefor cancer cell growth. Relevant features of the Survivin gene includeits developmentally- and differentiation-regulated expression, itsnearly identical and complementary DNA sequence with the factor Xareceptor EPR-1, and its abundant in vivo expression in common humanmalignancies, but not in the adjacent non-neoplastic population. Asdescribed below, targeting Survivin expression bymetallothionein-induction of EPR-1 mRNA resulted in apoptosis andinhibition of proliferation of HeLa cell transfectants.

[0044] In addition to their contribution to hemostasis, cellularreceptors for blood proteases have recently emerged as pleiotropicsignaling molecules, playing a crucial role in embryologic development(Connolly, A. J. et al., Nature (1996) 381:516-519), and vasculogenesis(Carmeliet, P. et al., Nature (1996) 383:73-75). In this context, theSurvivin gene was isolated by hybridization with the cDNA for EPR-1, areceptor for factor Xa contributing to procoagulant activity (Altieri,D. C., FASEB J (1995) 9:860-865), and T cell activation (Duchosal, M. A.et al., Nature (1996) 380:352-356). Although the Survivin codingsequence was found to be nearly identical to the EPR-1 cDNA, itsorientation was unambiguously assigned to the antisense EPR-1 strand forthe position of the consensus splice sites at intron-exon boundaries(Padgett, R. A. et al., Ann Rev Biochem (1986) 55:1119-1150). On theother hand, the authenticity of the EPR-1 “sense” strand wasdemonstrated in previous studies, when mammalian cells transfected withthe EPR-1 cDNA or with chimeric EPR-1 constructs (Ambrosini, G. et al.,J Biol Chem (1996) 271:1243-1248 and Altieri, D. C., FASEB J (1995)9:860-865), were recognized by anti-EPR-1 mAbs and bound factor Xa in aspecific and saturable reaction.

[0045] These findings could be reconciled by the existence of multiple,highly homologous, EPR-1 transcripts oriented in opposite directions.The heterogeneity of EPR-1 mRNA and the complex pattern of Southernhybridization support this hypothesis. Previously, double strand EPR-1probes detected three strongly hybridizing bands of 1.9, 3.4 and ˜1.5 kbin mRNA of EPR-1⁺ cells (Altieri, D. C., FASEB J (1995) 9:860-865).Here, single strand-specific probes confirmed the presence of multiplemature and polyadenylated EPR-1-related messages, and revealed that the1.9 and 3.4 kb bands corresponded to two highly regulated, antisenseEPR-1 transcripts, while the 1.5 kb band, more accurately defined as 1.2kb, coincided with a genuine EPR-1-encoding message. While the 1.9 kbantisense transcript clearly originated from the Survivin gene describedhere, a gene encoding the 1.2 kb “sense” EPR-1 message has not yet beenidentified.

[0046] However, (i) the presence of several genomic EPR-1-hybridizingbands unrelated to the Survivin gene, (ii) the different restrictionpattern of EPR-1 sequences in various species, and (iii) the numerousexpressed sequence tag database entries matching (P=0.018−7×10⁻¹¹) thepositive (accession n. W46267), or the negative (accession n. W34764,W83810, T29149) EPR-1 strand, altogether suggest the existence of atleast a second, highly-related, EPR-1 gene oriented in the oppositedirection to that described here, and encoding the previouslycharacterized factor Xa receptor (Altieri, D. C., FASEB J (1995)9:860-865).

[0047] A similar situation could arise from gene duplication event(s)involving EPR-1 sequences. Interestingly, the single hybridizationsignal detected on chromosome 17q25, and the single hybridizing bandsidentified in a Southern blot of high molecular weight genomic DNA,suggest that EPR-1-related sequences potentially oriented in oppositedirections may be adjacent in close proximity, within a physicalinterval of 75-130 kb.

[0048] The presence of multiple EPR-1 transcripts oriented in oppositedirections implies a reciprocal regulatory mechanism by naturallyoccurring antisense. This is consistent with the predominantlydiscordant and mutually exclusive distribution of sense and antisenseEPR-1 messages in developing or adult tissues in vivo, and during HL-60cell terminal differentiation. While antisense regulation is common inprokaryotes (Green, P. J. et al., Annu Rev Biochem (1986) 55:569-597), agrowing number of eukaryotic gene products have been recentlycharacterized for the occurrence of functional antisense transcriptspotentially participating in gene regulation, including basic fibroblastgrowth factor (Kimmelman, D. et al., Cell (1989) 59:687-696; Murphy, P.R. et al., Molecular Endocrinology (1994) 8:852-859), a1(I) collagen(Farrell, C. M. et al., J Biol Chem (1995) 270:3400-3408 and Lukens,1995), n-myc (Krystal, G. W. et al., Mol Cell Biol (1990) 10:4180-4191),c-myc (Celano, P. et al., J Biol Chem (1992) 267:15092-15096), p53(Khochbin, S. et al., EMBO J (1989) 8:4107-4114), c-erbAa (Lazar, M. A.et al., Mol Cell Biol (1989) 9:1128-1136), and CD3 ζ/η/θ locus (Lerner,A. et al., J Immunol (1993) 151:3152-3162).

[0049] As described below, the existence of a EPR-1/Survivin genebalance regulated by functional antisense was demonstrated in HeLa celltransfectants, when metallothionein-induced transcription of the EPR-1“sense” strand suppressed the expression of Survivin and profoundlyinfluenced apoptosis/cell proliferation (see below). This regulatorymechanism was not due to a potential protein association between EPR-1and Survivin, since the EPR-1 construct used for these experimentslacked a translational initiation codon. Additional experiments haveevaluated the ability of a Survivin antisense to inhibit cell growth.This was done by transiently co-transfecting the Survivin antisense witha lacZ reported plasmid and making a determination of cell viabilityafter a 48-h transfection in β-galactosidase expressing cells. Theresults indicated that the viability of Survivin antisense transfectantswas <20% of control cells transfected with the empty vector. A controlantisense of ICAM-1-(intercellular adhesion molecule-1) similarlyco-transfected in HeLa cells was ineffective.

[0050] Survivin was found to be a small protein of 142 amino acids(˜16.5 kDa) with no amino acid sequence homology to EPR-1, anddesignated Survivin for the presence of a BIR-homologous domain(Birnbaum, M. J. et al., J Virology (1994) 68:2521-2528; Clem, R. J. etal., Mol Cell Biol (1994) 14:5212-5222) found in IAP inhibitors ofapoptosis (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A.et al., Cell (1995) 83:1253-1262; Liston, P. et al., Nature (1996)379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252; Roy, N. et al.,Cell (1995) 80:167-178). Based on overall sequence conservation, theabsence of a carboxy terminus RING finger and the presence of a single,partially conserved, BIR domain, Survivin is the most distantly relatedmember of the IAP family, sharing the highest degree of similarity withNAIP (Roy, N. et al., Cell (1995) 80:167-178). Thus, unlike bcl-2 orother IAP proetins, Survivin is undetectable in adult tissues, butbecomes prominetnly expressed in all the most common human cancers oflung, colon, breast, pancreas, and prostate, and in ˜50% of high-gradenon-Hodgkin's lymphomas, in vivo. Additionally, unlike other IAPproteins (Deveraux, Q. et al., Nature (1997) 388:300-304), Survivin doesnot bind caspases in a cell-free system (Roy, N. et al., Blood (1997)595:2645.

[0051] Consistent with the anti-apoptosis properties of IAP proteins invitro (Duckett, C. S. et al., EMBO J (1996) 15:2685-2694; Liston, P. etal., Nature (1996) 379:349-353), and in vivo (Hay, B. A. et al., Cell(1995) 83:1253-1262), inhibition of Survivin expression by the EPR-1transcript (which naturally is antisense to Survivin) resulted inincreased apoptosis, as determined by in situ internucleosomal DNAfragmentation in HeLa cell transfectants. The ability of a RINGfinger-less IAP protein to counteract apoptosis is not without aprecedent, as demonstrated by the suppression of apoptosis mediated byNAIP (Liston, P. et al., Nature (1996) 379:349-353), and by the in vivogain-of-function of a Drosophila IAP protein following deletion of theRING finger (Hay, B. A. et al., Cell (1995) 83:1253-1262). Althoughanti-apoptosis genes are thought to play an indirect role in cellgrowth, by favoring the accumulation of oncogenic mutations(s) inaberrantly long-living cells (Reed, J. C., J Cell Biol (1994) 124:1-6),down-regulation of Survivin resulted in a profound inhibition of HeLacell proliferation. While this may derive from rapid disappearance ofHeLa cells expressing the highest levels of antisense transcripts byapoptosis, a similar decrease in tumor cell proliferation has beenreported in vivo after antisense inhibition of bcl-2 (Reed, J. C. etal., Proc Natl Acad Sci USA (1990) 87:3660-3664).

[0052] The possibility that IAP proteins may play a more general role incell proliferation, not exclusively restricted to apoptosis inhibition,has been proposed earlier. Rothe et al., have recently demonstrated thatthe amino terminus BIR in two IAP proteins (cIAPs) physically interactswith the signal transducers associated with the 75 kDa TNF receptor(Rothe, M. et al., Cell (1995) 83:1243-1252), a molecule primarilyimplicated in cell proliferation and survival rather than apoptoticsignaling (Tartaglia, L. A. et al., Immunol Today (1992) 13:151-153).While it is not known if Survivin is physically linked to signalingmolecules (Rothe, M. et al., Cell (1995) 83:1243-1252), the structuraldivergence of its BIR as compared with other IAP proteins (Duckett, C.S. et al., EMBO J (1996) 15:2685-2694; Hay, B. A. et al., Cell (1995)83:1253-1262; Liston, P. et al., Nature (1996) 379:349-353; Rothe, M. etal., Cell (1995) 83:1243-1252; Roy, N. et al., Cell (1995) 80:167-178),may confer specificity for supramolecular interaction(s) potentiallyrelevant to its particular mechanism of apoptosis inhibition/cellgrowth.

[0053] Dysregulation of programmed cell death (apoptosis) has recentlyemerged as a primary mechanism contributing to the pathogenesis ofvarious human diseases, including cancer (Steller, H., Science (1995)267:1445-1449; Thompson, C. B., Science (1995)267:1456-1462). While theimpact of anti-apoptosis gene(s) in neoplasia is highlighted by the roleof bcl-2 in follicular lymphoma (Korsmeyer, S. J., Blood (1992)80:879-886), a potential distribution of IAP proteins in cancer had notbeen previously investigated. In this context, one of the most strikingcharacteristics of Survivin was its abundant expression in activelyproliferating transformed cell lines, and in all the most common humanmalignancies of lung, colon, pancreas, and breast, in vivo, but not inthe non-neoplastic adjacent cell population. This distribution inmultiple human cancers may signal a fundamental role of this molecule inapoptosis/cell proliferation mechanisms in neoplasia. By analogy withthe paradigm of bcl-2, over-expression of Survivin in cancer may lead toaberrantly prolonged cell viability (Veis, D. J. et al., Cell (1993)75:229-240), increased resistance to chemotherapy-induced apoptosis(Miyashita, T. et al., Blood (1993) 81:151-157), and, as suggested bythe in vitro studies reported above, in a direct advantage fortransformed cell proliferation.

[0054] On the other hand, for its presence in normal PBMC and benignbreast adenomas, in vivo (unpublished observations), Survivin expressioncannot be interpreted per se as a marker of malignant transformation butmay reflect a more general, developmental- or cell type-specificresponse to certain stimuli. This is consistent with the presence ofSurvivin during normal embryonic (our unpublished observations) andfetal development, and its rapid disappearance in growth-arrested celltypes (i.e. vitamin D₃-treated HL-60), and terminally-differentiatedtissues, in vivo. At variance with other IAP proteins which areconstitutively found in adult mature tissues (Duckett, C. S. et al.,EMBO J (1996) 15:2685-2694; Liston, P. et al., Nature (1996)379:349-353; Rothe, M. et al., Cell (1995) 83:1243-1252), this patternof expression is reminiscent of the distribution of bcl-2 in fetaltissues (LeBrun, D. P. et al., Am J Pathol (1993) 142:743-753), and itsmore restricted presence in differentiated cells, correlating withsusceptibility to apoptosis (Hockenbery, D. M. et al., Proc Natl AcadSci USA (1991) 88:6961-6965).

[0055] In summary, these findings identify Survivin as a novel linkbetween IAP proteins and cancer, in vivo. A key implication of the datapresented below is the possibility to balance the effect of this potentanti-apoptosis gene by manipulating a normal cell regulatory mechanism,centered on the expression of EPR-1 (Altieri, D. C., FASEB J (1995)9:860-865). Targeting Survivin may then remove a selective advantage fortransformed cell growth and be therapeutically beneficial to increasethe susceptibility of cancer cells to chemotherapy-induced apoptosis.Along the same line, identification of polymorphic markers andconstruction of extended aplotypes within and around the EPR-1/Survivinlocus may provide new insights on the population genetics ofsusceptibility to chemotherapy.

[0056] III. Specific Embodiments

[0057] A. Survivin Protein

[0058] The present invention provides isolated Survivin protein, as wellas allelic variants of the Survivin protein, and conservative amino acidsubstitutions of the Survivin protein. As used herein, the Survivinprotein (or Survivin) refers to a protein that has the amino acidsequence of human Survivin depicted in FIG. 4. The term “Survivinprotein” also includes naturally occurring allelic variants of Survivin,naturally occurring proteins that have a slightly different amino acidsequence than that specifically recited above. Allelic variants, thoughpossessing a slightly different amino acid sequence than those recitedabove, will still have the requisite ability to inhibit cellularapoptosis.

[0059] As used herein, the Survivin family of proteins refers toSurvivin proteins that have been isolated from organisms in addition tohumans. The methods used to identify and isolate other members of theSurvivin family of proteins are described below.

[0060] Survivin is a member of the IAP (inhibitory apoptosis proteins)family of protein. However, Survivin is the first member of a uniquesubfamily of IAP proteins that differ from other IAP proteins insignificant ways. Despite homology and sequence conservation in the BIRmodule between Survivin and other members of this gene family, there areimportant structural differences that are unique to members of theSurvivin family of proteins. First unlike any other IAP protein,Survivin has only one BIR module (most of the other molecules have 2-3).Further, Survivin does not contain a carboxy-terminal RING finger buthas a predicted coiled-coil instead. Only the Neuronal ApoptosisInhibitory Protein (NAIP) in the IAP family lacks a RING finger, butdoes not contain a carboxy-terminus coiled coil. Finally there is no DNAsequence similarity between Survivin and other IAP proteins (PCR primersdesigned on Survivin are unlikely to detect other IAP proteins andvice-versa).

[0061] The Survivin proteins of the present invention are preferably inisolated from. As used herein, a protein is said to be isolated whenphysical, mechanical or chemical methods are employed to remove theSurvivin protein from cellular constituents that are normally associatedwith the Survivin protein. A skilled artisan can readily employ standardpurification methods to obtain an isolated Survivin protein.

[0062] The Survivin proteins of the present invention further includeconservative variants of the Survivin proteins herein described. As usedherein, a conservative variant refers to alterations in the amino acidsequence that do not adversely affect the ability of the Survivinprotein to bind to a Survivin binding partner and/or to inhibit cellularapoptosis. A substitution, insertion or deletion is said to adverselyaffect the Survivin protein when the altered sequence prevents theSurvivin protein from associating with a Survivin binding partner and/orprevents the Survivin protein from inhibiting cellular apoptosis. Forexample, the overall charge, structure or hydrophobic/hydrophilicproperties of Survivin can be altered without adversely affecting theactivity of Survivin. Accordingly, the amino acid sequence of Survivincan be altered, for example to render the peptide more hydrophobic orhydrophilic, without adversely affecting the activity of Survivin.

[0063] The allelic variants, the conservative substitution variants andthe members of the Survivin family of proteins, will have the ability toinhibit cellular apoptosis. Such proteins will ordinarily have an aminoacid sequence having at least about 75% amino acid sequence identitywith the human Survivin sequence, more preferably at least about 80%,even more preferably at least about 90%, and most preferably at leastabout 95%. Identity or homology with respect to such sequences isdefined herein as the percentage of amino acid residues in the candidatesequence that are identical with the known peptides, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and including any conservative substitutions as beinghomologous. N-terminal, C-terminal or internal extensions, deletions, orinsertions into the peptide sequence shall not be construed as affectinghomology.

[0064] Thus, the Survivin proteins of the present invention includemolecules having the amino acid sequences disclosed in FIG. 1; fragmentsthereof having a consecutive sequence of at least about 3, 5, 10 or 15amino acid residues of the Survivin protein; amino acid sequencevariants of such sequence wherein an amino acid residue has beeninserted N- or C-terminal to, or within, the disclosed Survivinsequence; amino acid sequence variants of the disclosed Survivinsequence, or their fragments as defined above, that have beensubstituted by another residue. Contemplated variants further includethose containing predetermined mutations by, e.g., homologousrecombination, site-directed or PCR mutagenesis, and the correspondingSurvivin proteins of other animal species, including but not limited torabbit, rat, murine, porcine, bovine, ovine, equine and non-humanprimate species, and the alleles or other naturally occurring variantsof the Survivin family of proteins; and derivatives wherein the Survivinprotein has been covalently modified by substitution, chemical,enzymatic, or other appropriate means with a moiety other than anaturally occurring amino acid (for example a detectable moiety such asan enzyme or radioisotope). The recombinant Survivin protein also can beused to solve the molecular structure of Survivin by 2D-NMR, circulardichroism and X-ray crystallography, thus integrating the site-directedmutagenesis approach and the rational design of specific small moleculeinhibitors.

[0065] As described below, members of the Survivin family of proteinscan be used: 1) as a target to block Survivin mediated inhibition ofcellular apoptosis, 2) to identify and isolate binding partners thatbind Survivin, 3) in methods to identify agents that block theassociation of Survivin with a Survivin binding partner, 4) as a targetto assay for Survivin mediated inhibition of cellular apoptosis, 5) asan agent to block cellular apoptosis, administered alone or as part of acombination therapy, 6) as a binding partner in an assay to quantitatecirculating levels of anti-Survivin antibodies, 7) as an antigen toelicit production of anti-Survivin antibodies that in turn can be usedin an an assay to quantitate circulating levels of Survivin and or canbe used for immunohistochemical purposes, and 8) as a therapeuticanti-cancer vaccine, or component of a polyvalent vaccine.

[0066] B. Anti-Survivin Antibodies

[0067] The present invention further provides antibodies thatselectively bind to a Survivin protein. The anti-Survivin antibodiesparticularly contemplated include monoclonal and polyclonal antibodiesas well as fragments containing the antigen binding domain and/or one ormore complement determining regions.

[0068] Antibodies are generally prepared by immunizing a suitablemammalian host using a Survivin protein, or fragment, in isolated orimmunoconjugated form (Harlow, Antibodies, Cold Spring Harbor Press, NY(1989)). FIG. 9 provides a Jameson-Wolf plot of the antigenic index ofvarious regions of Survivin. Such regions, in combination with the otherstructural analysis provided in FIG. 9, provide suitable fragments foruse in generating Survivin specific antibodies. Methods for preparingimmunogenic conjugates of a protein with a carrier such as BSA, KLH, orother carrier proteins are well known in the art. In some circumstances,direct conjugation using, for example, carbodiimide reagents may beused; in other instances linking reagents such as those supplied byPierce Chemical Co., Rockford, Ill., may be effective.

[0069] Administration of the Survivin immunogen is conducted generallyby injection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

[0070] While the polyclonal antisera produced in this way may besatisfactory for some applications, for pharmaceutical compositions,monoclonal antibody preparations are preferred. Immortalized cell lineswhich secrete a desired monoclonal antibody may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the Survivin peptide.When the appropriate immortalized cell culture secreting the desiredantibody is identified, the cells can be cultured either in vitro or byproduction in ascites fluid.

[0071] The desired monoclonal antibodies are then recovered from theculture supernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

[0072] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of receptor can also be produced in the context ofchimeras or CDR grafted antibodies of multiple species origin.

[0073] The antibodies thus produced are useful not only as modulators ofthe association of Survivin with a Survivin binding partner, but arealso useful in immunoassays for detecting Survivin expression/activityand for the purification of Survivin and associated binding partners.

[0074] C. Survivin Encoding Nucleic Acid Molecules

[0075] The present invention further provides nucleic acid moleculesthat encode Survivin, and the related Survivin proteins hereindescribed, preferably in isolated form For convenience, all Survivinencoding nucleic acid molecules will be referred to as the Survivinencoding nucleic acid molecule, the Survivin gene, or Survivin. As usedherein, “nucleic acid” is defined as RNA or DNA that encodes a peptideas defined above, or is complementary to a nucleic acid sequenceencoding such peptides, or hybridizes to such a nucleic acid and remainsstably bound to it under stringent conditions, or encodes a polypeptidesharing at least 75% sequence identity, preferably at least 80%, andmore preferably at least 85%, with the peptide sequences. Specificallycontemplated are genomic DNA, cDNA, mRNA and antisense molecules, aswell as nucleic acids based on an alternative backbone or includingalternative bases whether derived from natural sources or synthesized.Such hybridizing or complementary nucleic acid, however, is definedfurther as being novel and unobvious over any prior art nucleic acidincluding that which encodes, hybridizes under appropriate stringencyconditions, or is complementary to a nucleic acid encoding a Survivinprotein according to the present invention.

[0076] As used herein, “stringent conditions” are conditions in whichhybridization yields a clear and detectable sequence. Stringentconditions are those that (1) employ low ionic strength and hightemperature for washing, for example, 0.015M NaCl/0.0015M sodiumtitrate/0.1% SDS at 50° C., or (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC and 0.1% SDS. A skilled artisan can readily determineand vary the stringency conditions appropriately to obtain a clear anddetectable hybridization signal.

[0077] As used herein, a nucleic acid molecule is said to be “isolated”when the nucleic acid molecule is substantially separated fromcontaminant nucleic acid encoding other polypeptides from the source ofnucleic acid.

[0078] The present invention further provides fragments of the Survivinencoding nucleic acid molecule. As used herein, a fragment of a Survivinencoding nucleic acid molecule refers to a small portion of the entireprotein encoding sequence. The size of the fragment will be determinedby the intended use. For example, if the fragment is chosen so as toencode an active portion of the Survivin protein, such as the C-terminalβ coils or the IAP motif, the fragment will need to be large enough toencode the functional region(s) of the Survivin protein. If the fragmentis to be used as a nucleic acid probe or PCR primer, then the fragmentlength is chosen so as to obtain a relatively small number of falsepositives during probing/priming. FIG. 1 identifies fragments of theSurvivin gene that are particularly useful as selective hybridizationprobes or PCR primers.

[0079] Fragments of the Survivin encoding nucleic acid molecules of thepresent invention (i.e., synthetic oligonucleotides) that are used asprobes or specific primers for the polymerase chain reaction (PCR), orto synthesize gene sequences encoding Survivin proteins can easily besynthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci, et al., J Am Chem Soc (1981) 103:3185-3191 or usingautomated synthesis methods. In addition, larger DNA segments canreadily be prepared by well known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the Survivingene, followed by ligation of oligonucleotides to build the completemodified Survivin gene.

[0080] The Survivin encoding nucleic acid molecules of the presentinvention may further be modified so as to contain a detectable labelfor diagnostic and probe purposes. As described above such probes can beused to identify other members of the Survivin family of proteins and asdescribed below, such probes can be used to detect Survivin expressionand tumor growth potential. A variety of such labels are known in theart and can readily be employed with the Survivin encoding moleculesherein described. Suitable labels include, but are not limited to,biotin, radiolabeled nucleotides and the like. A skilled artisan canemploy any of the art known labels to obtain a labeled Survivin encodingnucleic acid molecule.

[0081] Since the Survivin gene is an antisense or reverse orientation ofthe EPR-1 gene, particularly preferred are single-stranded probes foruse in diagnostic purposes. Specifically, single-stranded diagnosticprobes can be used to selectively hybridize to mRNA that encodesSurvivin. Single-stranded probes can be generated using known methods inwhich one strand of a double-stranded probe is isolated or in which asingle stranded RNA probe is generated.

[0082] Modifications to the primary structure itself by deletion,addition, or alteration of the amino acids incorporated into the proteinsequence during translation can be made without destroying the activityof the protein. Such substitutions or other alterations result inproteins having an amino acid sequence encoded by DNA falling within thecontemplated scope of the present invention.

[0083] D. Isolation of Other Survivin Encoding Nucleic Acid Molecules

[0084] As described above, the identification of the human Survivinencoding nucleic acid molecule allows a skilled artisan to isolatenucleic acid molecules that encode other members of the Survivin familyof proteins in addition to the human sequence herein described.

[0085] Essentially, a skilled artisan can readily use the amino acidsequence of Survivin to generate antibody probes to screen expressionlibraries prepared from cells. Typically, polyclonal antiserum frommammals such as rabbits immunized with the purified Survivin protein (asdescribed below) or monoclonal antibodies can be used to probe amammalian cDNA or genomic expression library, such as lambda gtlllibrary, to obtain the appropriate coding sequence for Survivin, orother members of the Survivin family of proteins. The cloned cDNAsequence can be expressed as a fusion protein, expressed directly usingits own control sequences, or expressed by constructions using controlsequences appropriate to the particular host used for expression of theenzyme. FIG. 1 identifies important antigenic and/or putative operativedomains found in the Survivin protein sequence. Such regions arepreferred sources of antigenic portions of the Survivin protein for theproduction of probe, diagnostic, and therapeutic antibodies.

[0086] Alternatively, a portion of the Survivin encoding sequence hereindescribed can be synthesized and used as a probe to retrieve DNAencoding a member of the Survivin family of proteins from any mammalianorganisms that contains such a protein. Oligomers containingapproximately 18-20 nucleotides (encoding about a 6-7 amino acidstretch) are prepared and used to screen genomic DNA or cDNA librariesto obtain hybridization under stringent conditions or conditions ofsufficient stringency to eliminate an undue level of false positives.

[0087] Additionally, pairs of oligonucleotide primers can be preparedfor use in a polymerase chain reaction (PCR) to selectively clone aSurvivin-encoding nucleic acid molecule. A PCR denature/anneal/extendcycle for using such PCR primers is well known in the art and canreadily be adapted for use in isolating other Survivin encoding nucleicacid molecules. FIG. 1 identifies regions of the human Survivin genethat are particularly well suited for use as a probe or as primers.

[0088] E. rDNA Molecules Containing a Survivin Encoding Nucleic AcidMolecule

[0089] The present invention further provides recombinant DNA molecules(rDNAs) that contain a Survivin encoding sequence. As used herein, arDNA molecule is a DNA molecule that has been subjected to molecularmanipulation in vitro. Methods for generating rDNA molecules are wellknown in the art, for example, see Sambrook et al., Molecular Cloning(1989). In the preferred rDNA molecules, a Survivin encoding DNAsequence is operably linked to expression control sequences and/orvector sequences.

[0090] The choice of vector and/or expression control sequences to whichone of the Survivin encoding sequences of the present invention isoperably linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed. A vector contemplated by the present inventionis at least capable of directing the replication or insertion into thehost chromosome, and preferably also expression, of the Survivin geneincluded in the rDNA molecule.

[0091] Expression control elements that are used for regulating theexpression of an operably linked protein encoding sequence are known inthe art and include, but are not limited to, inducible promoters,constitutive promoters, secretion signals, and other regulatoryelements. Preferably, the inducible promoter is readily controlled, suchas being responsive to a nutrient in the host cell's medium.

[0092] In one embodiment, the vector containing a Survivin encodingnucleic acid molecule will include a prokaryotic replicon, i.e., a DNAsequence having the ability to direct autonomous replication andmaintenance of the recombinant DNA molecule extrachromosomally in aprokaryotic host cell, such as a bacterial host cell, transformedtherewith. Such replicons are well known in the art. In addition,vectors that include a prokaryotic replicon may also include a genewhose expression confers a detectable marker such as a drug resistance.Typical bacterial drug resistance genes are those that confer resistanceto ampicillin or tetracycline.

[0093] Vectors that include a prokaryotic replicon can further include aprokaryotic or viral promoter capable of directing the expression(transcription and translation) of the Survivin encoding gene sequencesin a bacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond,Calif.), pPL and pKK223 available from Pharmacia, Piscataway, N.J.

[0094] Expression vectors compatible with eukaryotic cells, preferablythose compatible with vertebrate cells, can also be used to form rDNAmolecules that contain a Survivin encoding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), the vector pCDM8 described herein, and the like eukaryoticexpression vectors.

[0095] Eukaryotic cell expression vectors used to construct the rDNAmolecules of the present invention may further include a selectablemarker that is effective in an eukaryotic cell, preferably a drugresistance selection marker. A preferred drug resistance marker is thegene whose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene. Southern et al., J Mol Anal Genet (1982)1:327-341. Alternatively, the selectable marker can be present on aseparate plasmid, and the two vectors are introduced by co-transfectionof the host cell, and selected by culturing in the appropriate drug forthe selectable marker.

[0096] F. Host Cells Containing an Exogenously Supplied SurvivinEncoding Nucleic Acid Molecule

[0097] The present invention flirter provides host cells transformedwith a nucleic acid molecule that encodes a Survivin protein of thepresent invention. The host cell can be either prokaryotic oreukaryotic. Eukaryotic cells useful for expression of a Survivin proteinare not limited, so long as the cell line is compatible with cellculture methods and compatible with the propagation of the expressionvector and expression of the Survivin gene product. Preferred eukaryotichost cells include, but are not limited to, yeast, insect and mammaliancells, preferably vertebrate cells such as those from a mouse, rat,monkey or human fibroblastic cell line, the most preferred being cellsthat do not naturally express a Survivin protein. Preferred eukaryotichost cells include the murine IL-3 dependent cell line BaF3, and thelike eukaryotic tissue culture cell lines.

[0098] Any prokaryotic host can be used to express a Survivin-encodingrDNA molecule. The preferred prokaryotic host is E. coli.

[0099] Transformation of appropriate cell hosts with a rDNA molecule ofthe present invention is accomplished by well known methods thattypically depend on the type of vector used and host system employed.With regard to transformation of prokaryotic host cells, electroporationand salt treatment methods are typically employed, see, for example,Cohen et al., Proc Natl Acad Sci USA (1972) 69:2110; and Maniatis etal., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982). With regard totransformation of vertebrate cells with vectors containing rDNAs,electroporation, cationic lipid or salt treatment methods are typicallyemployed, see, for example, Graham et al., Virol (1973) 52:456; Wigleret al., Proc Natl Acad Sci USA (1979) 76:1373-76.

[0100] Successfully transformed cells, i.e., cells that contain a rDNAmolecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrDNA of the present invention can be cloned to produce single colonies.Cells from those colonies can be harvested, lysed and their DNA contentexamined for the presence of the rDNA using a method such as thatdescribed by Southern, J Mol Biol (1975) 98:503, or Berent et al.,Biotech (1985) 3:208 or the proteins produced from the cell assayed viaan immunological method.

[0101] G. Production of Survivin Using a rDNA Molecule Encoding aSurvivin Protein

[0102] The present invention further provides methods for producing aSurvivin protein that uses one of the Survivin encoding nucleic acidmolecules herein described. In general terms, the production of arecombinant form of a Survivin protein typically involves the followingsteps.

[0103] First, a nucleic acid molecule is obtained that encodes aSurvivin protein, such as the nucleic acid molecule depicted in FIG. 1.If the Survivin encoding sequence is uninterrupted by introns, it isdirectly suitable for expression in any host. If not, then a splicedform of the Survivin encoding nucleic acid molecule can be generated andused or the intron containing nucleic acid molecule can be used in acompatible eukaryotic expression system.

[0104] The Survivin encoding nucleic acid molecule is then preferablyplaced in operable linkage with suitable control sequences, as describedabove, to form an expression unit containing the Survivin encodingsequences. The expression unit is used to transform a suitable host andthe transformed host is cultured under conditions that allow theproduction of the Survivin protein. Optionally the Survivin protein isisolated from the medium or from the cells; recovery and purification ofthe protein may not be necessary in some instances where some impuritiesmay be tolerated.

[0105] Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene and were discussed in detail earlier. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors. A skilled artisan can readily adapt any host/expressionsystem known in the art for use with Survivin encoding sequences toproduce a Survivin protein.

[0106] H. Inhibition of Cell Death Using Survivin

[0107] As provided above, Survivin has been shown to inhibit cellularapoptosis. Accordingly, Survivin can be used in methods to extend thelife of cells. In general, cellular apoptosis can be inhibited bycontacting a cell with Survivin.

[0108] The are a number of situation in which it is desirable to inhibitcellular apoptosis. For example, the death of cells in tissues andorgans being prepared for transport and transplant can be inhibitedusing the Survivin protein. Alternatively, cells lines can beestablished for long term culture using Survivin encoding nucleic acidmolecules expressed in the cell line.

[0109] Hence, Survivin protein or Survivin gene expression can be usedas a means to inhibit cellular apoptosis. In cell culture systems, theSurvivin protein can be introduced into a cell, for example vialiposomal, Penetrin-1 delivery, or inclusion in the cell growth media,to inhibit apoptosis. Alternatively, the Survivin gene can be introducedand expressed in cells to increase the longevity of cells in culture.These provide means and methods for increasing the ability of culturedcells to produce desired compounds as well as provide methods ofestablishing long-term culture of primary explants of cells and tissues.

[0110] In tissue transplant, typically tissues and organs are stored andtransported prior to transplant. Cell death, by mechanisms similar toapoptosis, can lead to the loss of viability of the tissues or organs.In this setting, infusion with Survivin protein can be used as a methodto inhibit cell death in such tissues and organs.

[0111] There are pathological conditions characterized by premature andunwanted cellular apoptosis, for example in accelerated aging disorders.It is already known that inactivating mutations in a IAP protein maycause human diseases. The example is for the NAIP (see above). Studiesof patients with SMA (Spinal muscular atrophy, a neurodegenrativedisease that is thought to be caused by aberrantly increased apoptosis)has demonstrated that the NAIP gene is inactivated and deleted in 75% ofthese patients (Roy et al. 1995, Cell 80:167). By extension,inactivating mutations in Survivin can result in degenerative diseasescharacterized by aberrantly increased cell death. Haplotypic markerswithin and around the Survivin locus on chromosome 17q25 can be used instudies of population genetics to determine if that locus has alreadybeen implicated in diseases with increased apoptosis. In such cases, theSurvivin gene or the Survivin protein can be used to treat theconditions. Accordingly, the Survivin protein, or a Survivin encodingnucleic acid molecule is administered to an individual as a means oftreating abnormal apoptosis.

[0112] I. Methods to Identify Survivin Binding Partners

[0113] Another embodiment of the present invention provides methods foruse in isolating and identifying binding partners of Survivin.Specifically, the Survivin protein can be used as a capture probe toidentify Survivin binding partners. As used herein, a Survivin bindingpartner is a biomolecule (such as a protein, DNA or other cofactor) thatbinds to Survivin and mediates Survivin inhibition of cellularapoptosis.

[0114] In detail, a Survivin protein is mixed with an extract orfraction of a cell that expresses Survivin under conditions that allowthe association of a binding partner with Survivin. After mixing,peptides that have become associated with Survivin are separated fromthe mixture. The binding partner that bound Survivin can then be removedand further analyzed.

[0115] To identify and isolate a binding partner, the entire Survivinprotein can be used. Alternatively, a fragment of a Survivin protein canbe used.

[0116] As used herein, a cellular extract refers to a preparation orfraction that is made from a lysed or disrupted cell. The preferredsource of cellular extracts will be cells that naturally expressSurvivin. Examples of such cells include, but are not limited to tumorcells and embryonic tissues.

[0117] A variety of methods can be used to obtain an extract of a cell.Cells can be disrupted using either physical or chemical disruptionmethods. Examples of physical disruption methods include, but are notlimited to, sonication and mechanical shearing. Examples of chemicallysis methods include, but are not limited to, detergent lysis and theenzyme lysis. In addition, the cellular extract can be prepared fromcells that have been freshly isolated from a subject or from cells orcell lines which have been cultured. A skilled artisan can readily adaptmethods for preparing cellular extracts in order to obtain extracts foruse in the present methods.

[0118] Once an extract of a cell is prepared, the extract is mixed withthe Survivin protein under conditions in which association of Survivinwith the binding partner can occur. A variety of conditions can be used,the most preferred being conditions that closely resemble conditionsfound in the cytoplasm of a Survivin-expressing cell. Features such asosmolarity, pH, temperature, and the concentration of cellular extractused, can be varied to optimize the association of the Survivin with thebinding partner.

[0119] After mixing under appropriate conditions, Survivin is separatedfrom the mixture. A variety of techniques can be utilized to separatethe mixture. For example, antibodies specific to Survivin can be used toimmunoprecipitate the Survivin and associated binding partner.Alternatively, standard chemical separation techniques such aschromatography and density/sediment centrifugation can be used.

[0120] After removal of nonassociated cellular constituents found in theextract, the binding partner can be dissociated from the Survivinprotein using conventional methods. For example, dissociation can beaccomplished by altering the salt concentration or pH of the mixture.

[0121] To aid in separating associated Survivin/binding partner pairsfrom the mixed extract, the Survivin protein can be immobilized on asolid support. For example, Survivin can be attached to a nitrocellulosematrix or acrylic beads. Attachment of Survivin to a solid supportfurther aids in separating peptide/binding partner pair from otherconstituents found in the extract.

[0122] Alternatively, the Survivin-encoding nucleic acid molecule can beused in a yeast two-hybrid system. The yeast two-hybrid system has beenused to identify other protein partner pairs and can readily be adaptedto employ the Survivin encoding molecules herein described.

[0123] J. Use of Survivin Binding Partners

[0124] Once isolated, the Survivin binding partners obtained using theabove described methods can be used for a variety of purposes. Thebinding partners can be used to generate antibodies that bind to theSurvivin binding partner using techniques known in the art. Antibodiesthat bind a Survivin binding partner can be used to assay Survivinactivity, as a therapeutic agent to modulate a biological orpathological process mediated by Survivin, or to purify the bindingpartner. These uses are described in detail below.

[0125] K. Methods to Identify Agents that Block Survivin/Binding PartnerInteractions

[0126] Another embodiment of the present invention provides methods foridentifying agents that reduce or block the association of Survivin witha Survivin binding partner. Specifically, Survivin is mixed with aSurvivin binding partner in the presence and absence of an agent to betested. After mixing under conditions that allow association of Survivinwith the Survivin binding partner, the two mixtures are analyzed andcompared to determine if the agent reduced or blocked the association ofSurvivin with the Survivin binding partner. Agents that block or reducethe association of Survivin with the Survivin binding partner will beidentified as decreasing the amount of association present in the samplecontaining the tested agent.

[0127] As used herein, an agent is said to reduce or blockSurvivin/Survivin binding partner association when the presence of theagent decreases the extent to which or prevents the Survivin bindingpartner from becoming associated with Survivin. One class of agents willreduce or block the association by binding to the Survivin bindingpartner while another class of agents will reduce or block theassociation by binding to Survivin.

[0128] The Survivin binding partner used in the above assay can eitherbe an isolated and fully characterized protein or can be a partiallycharacterized protein that binds to Survivin or a Survivin bindingpartner that has been identified as being present in a cellular extract.It will be apparent to one of ordinary skill in the art that so long asthe Survivin binding partner has been characterized by an identifiableproperty, e.g., molecular weight, the present assay can be used.

[0129] Agents that are assayed in the above method can be randomlyselected or rationally selected or designed. As used herein, an agent issaid to be randomly selected when the agent is chosen randomly withoutconsidering the specific sequences involved in the association of theSurvivin with the Survivin binding partner. An example of randomlyselected agents is the use a chemical library or a peptide combinatoriallibrary, or a growth broth of an organism.

[0130] As used herein, an agent is said to be rationally selected ordesigned when the agent is chosen on a nonrandom basis which takes intoaccount the sequence of the target site and/or its conformation inconnection with the agent's action. As described above, there are twosites of action for agents that block Survivin/Survivin binding partnerinteraction: the binding partner contact site on Survivin and theSurvivin contact site on the Survivin binding partner. Agents can berationally selected or rationally designed by utilizing the peptidesequences that make up the contact sites of the Survivin/Survivinbinding partner pair. For example, a rationally selected peptide agentcan be a peptide whose amino acid sequence is identical to the Survivincontact site on the Survivin binding partner. Such an agent will reduceor block the association of Survivin with the binding partner by bindingto the Survivin binding partner.

[0131] The agents of the present invention can be, as examples,peptides, small molecules, vitamin derivatives, as well ascarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents of the presentinvention. One class of agents of the present invention are peptideagents whose amino acid sequences are chosen based on the amino acidsequence of the Survivin protein.

[0132] The peptide agents of the invention can be prepared usingstandard solid phase (or solution phase) peptide synthesis methods, asis known in the art. In addition, the DNA encoding these peptides may besynthesized using commercially available oligonucleotide synthesisinstrumentation and produced recombinantly using standard recombinantproduction systems. The production using solid phase peptide synthesisis necessitated if non-gene-encoded amino acids are to be included.

[0133] Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the Survivin or Survivinbinding partner. As described above, antibodies are obtained byimmunization of suitable mammalian subjects with peptides, containing asantigenic regions, those portions of the Survivin or binding partner,intended to be targeted by the antibodies. Critical regions include thecontact sites involved in the association of the Survivin with theSurvivin binding partner.

[0134] As discussed below, the important minimal sequence of residuesinvolved in Survivin activity define a functional linear domain that canbe effectively used as a bait for two-hybrid screening andidentification of potential Survivin-associated molecules. Use of suchSurvivin fragments will significantly increase the specificity of thescreening as opposed to using the full length molecule or the entire BIRdomain and is therefore preferred. Similarly, this linear sequence canbe also used as an affinity matrix also to isolate Survivin bindingproteins using a biochemical affinity purification strategy.

[0135] L. Uses for Agents that Block the Association of Survivin with aSurvivin Binding Partner

[0136] As provided in the Background section, Survivin inhibits cellularapoptosis. Agents that reduce or block the interactions of Survivin witha Survivin binding partner can be used to modulate biological andpathologic processes associated with Survivin function and activity.

[0137] In detail, a biological or pathological process mediated bySurvivin can be modulated by administering to a subject an agent thatblocks the interaction of Survivin with a Survivin binding partner.

[0138] As used herein, a subject can be any mammal, so long as themammal is in need of modulation of a pathological or biological processmediated by Survivin. The term “mammal” is meant an individual belongingto the class Mammalia. The invention is particularly useful in thetreatment of human subjects.

[0139] As used herein, a biological or pathological process mediated bySurvivin or Survivin binding to a Survivin binding partner refers to thewide variety of cellular events mediated by Survivin. Pathologicalprocesses refer to a category of biological processes which produce adeleterious effect. For example, a pathological process mediated bySurvivin is the inhibition of cellular apoptosis in tumor cells. Thispathological process can be modulated using agents that reduce or blockSurvivin/Survivin binding partner association or block Survivinexpression.

[0140] As used herein, an agent is said to modulate a pathologicalprocess when the agent reduces the degree or severity of the process.For example, an agent is said to modulate tumor cell proliferation whenthe agent decrease the rate or extent of cell division.

[0141] M. Administration of Survivin or Agents that Affect SurvivinActivity

[0142] The agents of the present invention, whether they be agents thatblock Survivin/binding partner association or the Survivin protein, canbe administered via parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, or buccal routes.Alternatively, or concurrently, administration may be by the oral route.The dosage administered will be dependent upon the age, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. For example, totreat tumor cells as a means of blocking Survivin inhibition ofapoptosis, an agent that blocks Survivin expression or the interactionof Survivin with a binding partner, is administered systemically orlocally to the individual being treated. As described below, there aremany methods that can readily be adapted to administer such agents.

[0143] The present invention further provides compositions containingSurvivin or one or more agents that block Survivin/binding partnerassociation. While individual needs vary, a determination of optimalranges of effective amounts of each component is within the skill of theart. Typical dosages comprise 0.1 to 100 μg/kg body wt. The preferreddosages comprise 0.1 to 10 μg/kg body wt. The most preferred dosagescomprise 0.1 to 1 μg body wt.

[0144] In addition to the pharmacologically active agent, thecompositions of the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically for delivery to the siteof action. Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers. Liposomes canalso be used to encapsulate the agent for delivery into the cell.

[0145] The pharmaceutical formulation for systemic administrationaccording to the invention may be formulated for enteral, parenteral ortopical administration. Indeed, all three types of formulations may beused simultaneously to achieve systemic administration of the activeingredient.

[0146] Suitable formulations for oral administration include hard orsoft gelatin capsules, pills, tablets, including coated tablets,elixirs, suspensions, syrups or inhalations and controlled release formsthereof.

[0147] In practicing the methods of this invention, the compounds ofthis invention may be used alone or in combination, or in combinationwith other therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadministered alongwith other compounds typically prescribed for these conditions accordingto generally accepted medical practice, such as chemotherapeutic agents.

[0148] N. Combination Therapy

[0149] Survivin, as well as agents of the present invention thatmodulate Survivin activity, can be provided alone, or in combinationwith another agents that modulate a particular biological orpathological process. For example, an agent of the present inventionthat reduces Survivin inhibited apoptosis can be administered incombination with other anti-cancer agents in methods to control cancercell growth. Alternatively, Survivin can be administered with otherprotective agents as a means for reducing cellular apoptosis. As usedherein, two agents are said to be administered in combination when thetwo agents are administered simultaneously or are administeredindependently in a fashion such that the agents will act at the sametime.

[0150] Inhibition of Survivin activity/expression can be used incombination with conventional chemotherapies. The timing for using achemotherapeutic agent in combination with inhibiting Survivinactivity/expression depends upon chemotherapeutic agent used and thetumor cell type treated. Examples of chemotherapeutic agents that can beused in combination with agents the effect Survivin activity/expression,includes, but is not limited to alkylating agents, such ascyclophosphamide (CTX; cytoxan), chloranbucil (CHL; leukeran), cisplatin(CisP; platinol) busulfan (myleran), melphalan, carmustine (BCNU),streptozotocin, triethylenemelamine (TEM), mitomycin C., and the likealkylating agents; anti-metabolites, such as methotrexate (MTX),etoposide (VP16; vepesid) 6-mercaptopurine (6MP), 6-thiocguanine (6TG),cytarabine (Ara-C), 5-fluorouracil (5FU), dacarbazine DTIC), and thelike anti-metabolites; antibiotics, such as actinomycin D, doxorubicin(DXR; adriaamycin), daunorubicin (daunomycin), bleomycin, mitlramycinand the like antibiotics; alkaloids, such as vinca alkaloids such asvincristine (VCR), vinblastine, and the like; and other antitumoragents, such as taxol and taxol derivatives, the cytostatic agentsglucocorticoids such as dexamethasone (DEX; decadron) andcorticosteroids such as prednisone, nucleoside enzyme inhibitors such ashydroxyurea, amino acid depleting enzymes such as asparaginase, and thelike diverse antitumor agents.

[0151] The use of the cytotoxic agents described above inchemotherapeutic regimens is generally well characterized in the cancertherapy arts, and their use herein falls under the same considerationsfor monitoring tolerance and effectiveness and for controllingadministration routes and dosages, with some adjustments. For example,the actual dosages of the cytotoxic agents may vary depending upon thepatient's cultured cell response determined by using the presenthistoculture methods. Generally, the dosage will be reduced compared tothe amount used in the absence of agents the effect Survivinactivity/expression.

[0152] Typical dosages of an effective cytotoxic agent can be in theranges recommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

[0153] O. Methods for Identifying Survivin Expression andSurvivin-Mediated Inhibition of Apoptosis

[0154] The present invention further provides methods for identifyingcells involved in Survivin-mediated inhibition of apoptosis as well astechniques that can be applied to diagnose biological and pathologicalprocesses associated with Survivin activity, the progression of suchconditions, the susceptibility of such conditions to treatment and theeffectiveness of treatment for such conditions. Specifically,Survivin-mediated inhibition of apoptosis can be identified bydetermining whether the Survivin protein is expressed in a cell. Cellsexpressing Survivin are considered to be inhibited from natural cellularapoptosis.

[0155] A variety of immunological and nucleic acid techniques can beused to determine if the Survivin protein, or a Survivin encoding mRNA,is produced in a particular cell. In one example, an extract of cells isprepared. The extract is then assayed to determine whether Survivin isexpressed in the cell. The degree of expression provides a measurementof the degree of inhibition of apoptosis. An increase in expression is ameasurement of an increased inhibition of apoptosis.

[0156] The measurement of Survivin expression can be used as a markerfor a variety of purposes. In tumors, the present of Survivin expressioncorrelates with the proliferative potential of the tumor. In theExamples, it is shown that lymphomas display varying levels of Survivinexpression; lymphomas showing little to no Survivin expression are lowgrade lymphomas that can be effectively treated while lymphomas showinghigh levels of Survivin expression are high grade aggressive lymphomasthat typically cannot be effectively treated. Accordingly, the level ofSurvivin expression in a lymphoma, or other tumor, can be used as apredictive measurement of the aggressiveness and treatability of thetumor: the higher the level of Survivin expression, the higher theaggressiveness of the tumor and the more difficult the treatment willbe.

[0157] For example, to determine a tumor's proliferative potential oreasy/prognosis of treatment, an extract is made of the tumor cells andthe extract is then analyzed, for example, by gel electrophoresis, todetermine whether a Survivin protein is present. The presence and levelof Survivin correlates with the proliferative potential of the cancerand the ease of treatment. Alternatively, as described above,single-strand probes can be used to identify Survivin-encoding mRNA inthe cellular extracts.

[0158] In addition to being a marker of tumor aggressiveness andtreatment potential, Survivin expression can be used as a measurement ofthe effectiveness of anti-tumor therapy. In the Examples, it is shownthat HL-60, a promylocytic cell line, had high levels of Survivinexpression. Treatment of HL-60 cells with retenoic acid, and anti-canceragent that acts by causing the differention of tumor cells, resulted ina reduction and elimination of Survivin expression. The reduction inexpression correlated with the degree of differentiation, the greaterthe differentiation, the lower the level of Survivin expression.Accordingly, Survivin expression can be used to measure theeffectiveness of anti-tumor treatment: if Survivin expression decreasesduring treatment, the treatment protocol is effective and can becontinued, whereas if Survivin expression remains unaltered, a differenttherapeutic regime or protocol needs to be performed.

[0159] P. Other Methods to Control Survivin Expression

[0160] The present invention further provides additional methods thatcan be used to control Survivin expression in a cell. As discussed aboveand below, the Survivin promoter has a CPG island upstream from itspromoter. CPG islands are known targets for DNA methylation. The DNAmethylation sites in the CPG island serves as a means for regulatingSurvivin expression: methylation of CPG islands results in thesuppression of transcription of the gene found downstream from thepromoter. Accordingly, agents that methylate DNA, such as DNA methylase,and agents that stimulate the production of endogenous methylases, canbe used to control Survivin expression. Specifically, Survivinexpression in a cell can be reduced or eliminated by causing the cell toincrease the level of DNA methylation, particularly at the CPG islandfound upstream from the Survivin gene.

[0161] In another method, Survivin expression can be reduced byincreasing the level of EPR-1 expression. As shown in the Examples,Survivin expression and EPR-1 expression are generally mutuallyexclusive, expression of EPR-1 results in a decrease or elimination ofSurvivin expression and visa-a-versa Accordingly, Survivin expressioncan be reduced by causing a cell to increase EPR-1 expression.

[0162] Q. Animal Models

[0163] We have isolated almost the complete structure of the mouseSurvivin gene. The gene is very conserved with its human counterpartincluding sizes of introns, exons and intron-exon boundaries. The codingregions of the mouse Survivin gene are 88%, to the extent sequenced,identical to the human protein, thereby demonstrating strongevolutionary conservation. We have also determined the differential anddevelopmentally-regulated distribution of Survivin during both human andmouse development. The availability of the complete structure of themouse Survivin gene and protein will allow the preparation of targetingvectors for gene knockout experiments and a more rational approach forthe generation of transgenic mice expressing Survivin under the controlof tissue-specific promoters.

[0164] The Survivin gene and the Survivin protein can serve as a targetfor gene therapy in a variety of contexts. For example, in oneapplication, Survivin-deficient non-human animals can be generated usingstandard knock-out procedures to inactivate a Survivin gene or, if suchanimals are non-viable, inducible Survivin antisense molecules can beused to regulate Survivin activity/expression. Alternatively, an animalcan be altered so as to contain a Survivin or antisense-Survivinexpression unit that directs the expression of Survivin or the antisensemolecule in a tissue specific fashion. In such a uses, a non-humanmammal, for example a mouse or a rat, is generated in which theexpression of the Survivin gene is altered by inactivated or activation.This can be accomplished using a variety of art-known procedures such astargeted recombination. Once generated, the Survivin-deficient animal,the animal that expresses Survivin in a tissue specific manner, or ananimal that expresses an antisense molecule can be used to 1) identifybiological and pathological processes mediated by Survivin, 2) identifyproteins and other genes that interact with Survivin, 3) identify agentsthat can be exogenously supplied to overcome Survivin deficiency and 4)serve as an appropriate screen for identifying mutations within Survivinthat increase or decrease activity.

[0165] For example, it is possible to generate transgenic miceexpressing the human minigene for Survivin in a tissue specific-fashionand test the effect of over-expression of the protein in district thatnormally do not contain Survivin. This strategy has been successfullyused for another family of apoptosis inhibitors, namely bcl-2 (Veis etal., Cell (1993) 75:229). Such an approach can readily be applied to theSurvivin protein and can be used to address the issue of a potentialbeneficial effect of Survivin in a specific tissue area to protect cellsfrom apoptosis (transplant).

[0166] R. Survivin Gene Therapy

[0167] In another embodiment, genetic therapy can be used as a means formodulating a Survivin-mediated biological or pathological processes. Forexample, in tumor therapy, it may be desirable to introduce into thesubject being treated a genetic expression unit that encodes a modulatorof Survivin expression, such as an antisense encoding nucleic acidmolecule. Such a modulator can either be constitutively produced orinducible within a cell or specific target cell. This allows a continualor inducible supply of a modulator of Survivin expression within thesubject. Blocking Survivin expression allows for the control of tumorcell growth. Similarly, cells may be genetically engineered to expressSurvivin, e.g., in allograft pancreatic β cells for transplantation.

[0168] The level of Survivin gene expression may correlate with thelevel of resistance to apoptosis. Thus, Survivin genes also find use inanti-apoptosis gene therapy. In particular, a functional Survivin genemay be used to sustain neuronal cells that undergo apoptosis in thecourse of a neurodegenerative disease, lymphocytes (i.e., T cells and Bcells), or cells that have been injured by ischemia.

[0169] Retroviral vectors, adenoviral vectors, adeno- associated viralvectors, or other viral vectors with the appropriate tropism for cellslikely to be involved in apoptosis (for example, epithelial cells) maybe used as a gene transfer delivery system for a therapeutic Survivingene construct. Numerous vectors useful for this purpose are generallyknown (Miller, Human Gene Therapy 15-14, 1990; Friedman, Science244:1275-1281, 1989; Eglitis and Anderson, Bio Techniques 6:608-614,1988; Tolstoshev and Anderson, current opinion in biotechnology 1:55-61,1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., NucleicAcid Research and Molecular Biology 36:311-322, 1987; Anderson, Science226:401-409, 1984; Moen, blood Cells 17:407-416, 1991; Miller et al.,Biotechniques 7:980-990, 1989; Le Gal La Salle et al., Science259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviralvectors are particularly well developed and have ben used in clinicalsettings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson etal., U.S. Pat. No. 5,399,346).

[0170] Non-viral approaches may also be employed for the introduction oftherapeutic DNA into cells otherwise predicted to undergo apoptosis. Forexample, Survivin may be introduced into a neuron or a T cell bylipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987;Ono et al., Neurosci. Lett. 117:259, 190; Brigham et al., Meth. Enz.101:512, 1983), asialorosonucoid-polylysine conjugation (Wu et al., J.Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989);or, less preferably, microinjection under surgical conditions (Wolff etal., Science 247:1465, 1990).

[0171] For any of the methods of application described above, thetherapeutic Survivin nucleic acid construct is preferably applied to thesite of the predicted apoptosis event (for example, by injection).However, it may also be applied to tissue in the vicinity of thepredicted apoptosis event or to a blood vessel supplying the cellspredicted to undergo apoptosis.

[0172] In the constructs described, Survivin cDNA expression can bedirected from any suitable promoter (e.g., the human cytomegalovirus(CMV), simian virus 40 (SV40), or metallothionein promoters), andregulated by any appropriate mammalian regulatory element. For example,if desired, enhancers known to preferentially direct gene expression inneural cells, T cells, or B cells may be used to direct Survivinexpression. The enhancers used could include, without limitation, thosethat are characterized as tissue- or cell-specific in their expression.Alternatively, if a Survivin genomic clone is used as a therapeuticconstruct (for example, following its isolation by hybridization withthe Survivin cDNA described above), regulation may be mediated by thecognate regulatory sequences or, if desired, by regulatory sequencesderived from a heterologous source, including any of the promoters orregulatory elements described above.

[0173] S. Use of the Survivin Promoter to Direct Gene Expression

[0174] The present invention further provides the promoter of theSurvivin gene in a form that can be used in generating expressionvectors. Specifically, the Survivin promoter, identified as being 5′from the ATG start codon in of Survivin, can be used to direct theexpression of an operably linked protein encoding DNA sequence. Sincethe Survivin promoter does not have a TATA box, a skilled artisan woulduse a 5′ fragment, such as nucleotides 2560-2920 (including exon 1). TheSurvivin promoter is expressed in fetal tissues and can therefore beused to target protein expression in specific cell types during specificstages of development. As discussed below, transfection of 3T3 cellswith the c-myc oncogene results in the up-regulation of Survivin mRNA asdetected by Northern blots. Accordingly, DNA encoding anti-tumorpolypeptides under the control of the Survivin promoter could be used totransfect tumor cell where they would be expressed. A skilled artisancan readily use the Survivin promoter in expression vectors usingmethods known in the art.

[0175] T. Preventative Anti-Apoptotic Therapy

[0176] In a patient diagnosed to be heterozygous for a Survivin mutationor to be susceptible to Survivin mutations (even if those mutations donot yet result in alteration or loss of Survivin biological activity),or a patient diagnosed with a degenerative disease (e.g., motor neurondegenerative diseases such as SMA or ALS diseases), or diagnosed as HIVpositive, any of the disclosed therapies may be administered before theoccurrence of the disease phenotype. For example, the therapies may beprovided to a patient who is HIV positive but does not yet show adiminished T cell count or other overt signs of AIDS. In particular,compounds shown to increase Survivin expression or Survivin biologicalactivity may be administered by any standard dosage and route ofadministration. Alternatively, gene therapy using a Survivin expressionconstruct may be undertaken to reverse or prevent the cell defect priorto the development of the degenerative disease.

[0177] The methods of the instant invention may be used to reduce ordiagnose the disorders described herein in any mammal, for example,humans, domestics pets, or livestock. Where a non-human mammal istreated or diagnosed, the Survivin polypeptide, nucleic acid, orantibody employed is preferably specific for that species.

[0178] U. Examples of Additional Apoptosis Assays

[0179] In addition to the foregoing discussion, specific examples ofapoptosis assays are also provided in the following references. Assaysfor apoptosis in lymphocytes are disclosed by: Li et al., “Induction ofapoptosis in uninfected lymphocytes by HIV-1 Tat protein”, Science268:429-431, 1995; Gibellini et al., “Tat-expressing Jurkat cells showan increased resistance to different apoptotic stimuli, including acutehuman immunodeficiency virus-type 1 (HIV-1) infection”, Br. J. Haematol.89:24-33, 1995; Martin et al., “HIV-1 infection of human CD4⁺ T cells invitro. Differential induction of apoptosis in these cells.” J. Immunol.152:330-42, 1994; Terai et at., “Apoptosis as a mechanism of cell deathin cultured T lymphoblasts acutely infected with HIV-1”, J. Clin Invest.87:1710-5, 1991; Dhein et al., “Autocrine T-cell suicide mediated byAPO-1/(Fas/CD95)11, Nature 373:438-441, 1995; Katsikis et al., “Fasantigen stimulation induces marked apoptosis of T lymphocytes in humanimmunodeficiency virus-infected individuals”, J. Exp. Med.1815:2029-2036, 1995; Westendorp et al., Sensitization of T cells toCD95-mediated apoptosis by HIV-1 Tat and gp120”, Nature 375:497, 1995;DeRossi et al., Virology 198:234-44, 1994.

[0180] Assays for apoptosis in fibroblasts are disclosed by: Vossbeck etal., “Direct transforming activity of TGF-beta on rat fibroblasts”, Int.J. Cancer 61:92-97, 1995; Goruppi et al., “Dissection of c-myc domainsinvolved in S phase induction of HIH3T3 fibroblasts”, Oncogene9:1537-44, 1994; Fernandez et al., “Differential sensitivity of normaland Ha-ras transformed C3H mouse embryo fibroblasts tumor necrosisfactor; induction of bcl-2, c-myc, and manganese superoxide dismutase inresistant cells”, Oncogene 9:2009-17, 1994; Harrington et al., “cMyc-induced apoptosis in fibroblasts is inhibited by specificcytokines′, EMBO J., 13:3286-3295, 1994; Itoh et al., “A novel proteindomain required for apoptosis. Mutational analysis of human Fasantigen”, J. Biol. Chem. 268:10932-7, 1993.

[0181] Assays for apoptosis in neuronal cells are disclosed by: Melinoet al., “Tissue transglutaminase and apoptosis: sense and antisensetransfection studies with human neuroblastoma cells”, Mol. Cell Biol.14:6584-6596, 1994; Rosenblaum et al., “Evidence for hypoxia-induced,programmed cell death of cultured neurons”, Ann. Neurol. 36:864-870,1994; Sato et al., “Neuronal differentiation of PC12 cells as a resultof prevention of cell death by bcl-2”, J. Neurobiol. 25:1227-1234, 1994;Ferrari et al., “N-acetylcysteine D- and L-stereoisomers preventsapoptotic death of neuronal cells”, J. Neurosci. 1516:2857-2866, 1995;Talley et al., “Tumor necrosis factor alpha-induced apoptosis in humanneuronal cells: protection by the antioxidant N-acetylcysteine and thegenes bcl-2 and crma”, Mol. Cell Biol. 1585:2359-2366, 1995; Talley etal., “Tumor Necrosis Factor Alpha-Induced Apoptosis in Human NeuronalCells: Protection by the Antioxidant N-Acetylcysteine and the Genesbcl-2 and crma”, Mol. Cell. Biol. 15:2359-2366, 1995; Walkinshaw et al.,“Induction of apoptosis in catecholaminergic PC12 cells by L-DOPA.Implications for the treatment of Parkinson's disease,” J. Clin. Invest.95:2458-2464, 1995.

[0182] Assays for apoptosis in insect cells are disclosed by: Clem etal., “Prevention of apoptosis by a baculovirus gene during infection oninsect cells”, Science 254:1388-90, 1991; Crook et al., “Anapoptosis-inhibiting baculovirus gene with a zinc finger-like motif”, J.Virol: 67:2168-74, 1993; Rabizadeh et al., “Expression of thebaculovirus p35 gene inhibits mammalian neural cell death”, J.Neurochem. 61:2318-21, 1993; Birnbaum et al., “An apoptosis inhibitinggene from a nuclear polyhedrosis virus encoding a polypeptide withCys/His sequence motifs”, J. Virol. 68:2521-8, 1994; Clem et al., Mol.Cell. Biol. 14:5212-5222, 1994.

[0183] V. Use of Survivin in Tissue and Organ Transplantation Thepresent invention includes methods of inhibiting or preventing tissue ororgan transplant rejection in a subject, comprising the localadministration of a Survivin polypeptide, Survivin polypeptide fragment,an apoptosis,,inhibiting peptidomimetic thereof, a transgene encoding aSurvivin polypeptide or a transgene encoding a Survivin polypeptidefragment to the tissue, organ or to a site proximal to the transplant.Local delivery of the polypeptides, peptidomimetics to the tissue, organor to a site proximal to the transplant is accomplished by any meanscommonly available, including but not limited to direct local perfusion,injection, microsponges, microcapsules, liposomes or time-releaseddelivery vehicles.

[0184] Local delivery of a transgene encoding a Survivin polypeptide ora transgene encoding a Survivin polypeptide fragment to the tissue,organ or to a site proximal to the transplant may be accomplished withany available vector, via lipofection or via direct plasmid DNAinjection. See Qin et al. (1995) Transplantation 59(6): 809-816; LeCoultre et al. (1997) Eur. 3. Pediatr. Surg. 7(4):221-226; Wang et al.(1992) Transplantation 53(3):703-705; Wang et al. (1996) Transplantation61(12):1726-1729; Schmid et al., (1997) Eur. J Cardiothorac. Surg.11(6):1023-28; and Boasquevisque, C. et al. (1997) Ann. Thorac. Surg.63(6):1556-1561. Vectors encoding the transgene include both replicableand replication,,defective vectors, such as retroviral vectors,adenovirus vectors or other vectors with the appropriate tropism for thecells likely to be involved in apoptosis or cells proximal to the siteof apoptosis. In the transgene constructs, expression can be directedfrom any suitable promoter, including tissue specific promoters whichdirect gene expression in specific cell types, such as the human insulinpromoter. Local delivery of the transgene to the tissue, organ or to asite proximal to the transplant is accomplished by any means commonlyavailable, including but not limited to direct local perfusion,injection, microsponges, microcapsules, liposomes or time-releaseddelivery vehicles.

[0185] Without further description, it is believed that one of ordinaryskill in the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure. Other generic configurations will beapparent to one skilled in the art. All journal articles and otherpublished documents such as patents and patent applications are herebyincorporated by reference in their entireties.

EXAMPLES Example 1 Experimental procedures and cloning

[0186] Cells and cell culture. The following cell lines were obtainedfrom American Type Culture Collection (ATCC, Rockville, Md.),erythroleukemia HEL, B-lymphoma Daudi and JY, monocytic THP-1, Tleukemia Jurkat, epithelial carcinoma HeLa, promyelocytic HL-60, andnon-transformed human lung fibroblast Lu18. The T leukemia cell lineMOLT13 was characterized previously (Altieri, D. C., FASEB J (1995)9:860-865). Cells were maintained in culture in complete medium RPMI1640 or DMEM (HeLa, Lu18) (BioWhittaker, Walkersville, Md.),supplemented with 10% heat-inactivated fetal bovine serum (FBS,Whittaker), 2 mM L-glutamine, and 10 mM HEPES. Human umbilical veinendothelial cells (HUVEC) were isolated by collagenase treatment andmaintained in culture in DMEM medium supplemented with 20% FBS, 2 mML-glutamine and endothelial cell growth factor (Biomedical Technologies,Stoughton, Mass.).

[0187] Peripheral blood mononuclear cells (PBMC) were isolated fromheparinized blood collected from normal informed volunteers bydifferential centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway,N.J.) at 400 g for 22° C., and washed in phosphate buffered saline(PBS), pH 7.4. In some experiments, HL-60 cells were terminallydifferentiated to a mature monocytic phenotype by a 72 h culture in thepresence of 0.1 μM 1, 25-dihydroxy-vitamin D₃ and 17.8 μg/mlindomethacin (Sigma Chemical Co., St. Louis, Mo.). De novo induction ofdifferentiation-dependent markers on vitamin D₃-treated HL-60 cells,including CD11b/CD18 integrin (Hickstein, D. D. et al., J Immunol (1987)138:513-519) was determined by flow cytometry with anti-CD11b mAb LM2/1.

[0188] Genomic and cDNA cloning, chromosomal localization and Southernblots. A human P1 genomic library (Genome Systems, St. Louis, Mo.) wasscreened by hybridization with a 1.6 kb fragment containing the completehuman EPR-1 cDNA (Atieri, D. C., FASEB J (1995) 9:860-865). Threeoverlapping clones were isolated, purified and confirmed by Southernhybridization with the EPR-1 cDNA. Hybridizing fragments generated byrestriction digest with BamHI, HindIII and XbaI (Boehringer Mannheim,Indianapolis, Ind.) were cloned in pBluescript (pBSKS⁻, Stratagene, SanDiego, Calif.) for further analysis. An overlapping contig spanning14796 bp from two EPR-1-hybridizing P1 clones was arrayed, characterizedby restriction analysis, and completely sequenced on both strands by TaqFS polymerase-based automated sequencing using a Applied BioSystem Prism377 automated sequencer (Foster City, Calif.). In some experiments, 10mg of total RNA extracted from HeLa cells by the guanidiniumisothiocyanate method was primed with EPR-1 forward “sense”oligonucleotide C3/27 (bp 80-102) and reverse transcribed in thepresence of 200 U of Superscript II (Life Science, Grand Island, N.Y.)for 50 min at 42° C.

[0189] The resulting cDNA was amplified by PCR in the presence of 0.5 mgof EPR-1-derived primers T5/27 (bp 161-184) and G11/16 (1124-1098,numbering from the EPR-1 coding sequence), 200 mM dNTPs (New EnglandBiolabs, Beverly, Mass.) and 2 U Vent DNA polymerase (New EnglandBiolabs) in a total volume of 50 ml. After 35 cycles of amplificationwith annealing at 58° C. for 1 min, denaturation at 94° C. for 1 min andextension at 72° C. for 1 min, the product was analyzed by agarose gelelectrophoresis, subcloned in pCRII (Invitrogen Corp., San Diego,Calif.), and completely sequenced on both strands. Contig assembly, andDNA and protein sequence analyses were performed using Lasergene(DNASTAR, Madison, Wis.) and MacVector (Eastman Kodak, Rochester, N.Y.)software packages. Chromosomal location of the EPR-1-hybridizing genewas carried out by fluorescence in situ hybridization. Purified DNA froma EPR-1-hybridizing P1 clone was labeled with digoxigenin dUTP (AmershamCorp., Arlington Heights, Ill.) by nick translation.

[0190] The labeled probe was combined with sheared human DNA andhybridized to normal metaphase chromosomes derived fromphytohemagglutinin-stimulated PBMC in a solution containing 50%formamide, 10% dextran sulfate and 2×SSC. For two-color staining,biotin-conjugated probe D17Z1, specific for the centromere of chromosome17, was co-hybridized with the digoxigenin-labeled P1 clone. Specificstaining was detected by incubating the hybridized slides withfluoresceinated anti-digoxigenin antibodies and Texas red avidin. Slideswere counterstained with propidium iodide for one color labeling, orwith DAP1 for two color labeling. A total of 80 metaphase cells wereanalyzed with 69 cells exhibiting specific labeling. For Southernhybridization, human genomic DNA was extracted from HeLa cells accordingto published protocols, digested with EcoRI, BamHI, XbaI or HindIII,separated on a 0.8% agarose gel and transferred to GeneScreen nylonmembranes (New England Nuclear, Boston, Mass.).

[0191] After UV cross-linking (Stratalinker, Stratagene, San Diego,Calif.), the membrane was prehybridized with 100 mg/ml of denaturedsalmon sperm DNA (Promega Corp. Madison, Wis.) in 5×SSC, 0.5% SDS,5×Denhardt's solution and 0.1% sodium pyrophosphate at 65° C. in aroller hybridization oven (Hoefer Scientific, San Francisco, Calif.).Hybridization was carried out with gel-purified (GeneClean Bio101,Vista, Calif.), ³²P-dCTP (Amersham) random-primed labeled(Boehringer-Mannheim, Indianapolis, Ind.) 1.6 kb EPR-1 cDNA for 16 h at65° C.

[0192] After two washes in 2×SSC, 1% SDS for 30 min at 65° C., and0.2×SSC at 22° C., radioactive bands were visualized by autoradiographyusing a Kodak X-Omat AR X-ray film and intensifying screens (DuPont deNemours, Wilmington, Del.). In other experiments, culturedlymphoblastoid cells were embedded in LMP agarose (Bio Rad, Richmond,Calif.) at the concentration of 2×10⁶/220 μl block and DNA was extractedby standard procedures. After block digestion with MluI or NotI, sampleswere separated by pulsed field gel electrophoresis on a 1% agarose gelfor 20 h at 200 V with a pulse time of 75 sec using a Bio-Rad CHEF DRIIapparatus (Hercules, Calif.). After transfer to nylon membranes, and UVcross-linking, hybridization with the EPR-1 cDNA and washes were carriedout as described above.

[0193] In another series of experiments, a blot containing aliquots ofgenomic DNA isolated from several species (Clontech, San Francisco,Calif.) was hybridized with a 3′ 548 bp fragment of the EPR-1 cDNA, asdescribed above.

[0194] Northern blots. Single strand probes specific for sense orantisense EPR-1 sequences were generated by asymmetric PCR amplificationof a 301 bp fragment of the EPR-1 cDNA. The template, comprising thefirst 5′ 226 bp of the EPR-1 coding sequence plus 75 bp of the retainedregulatory intron (Altieri, D. C., FASEB J (1995) 9:860-865), wasgenerated by restriction digest of the EPR-1 cDNA with EcoRI (cloningsite) and SacII, gel-purified, and mixed in a total volume of 10 ml with15 pmol dNTP (New England Biolabs), 7.5 pmol dCTP, and 25 mCi ³²P-dCTP(Amersham), in the presence of 20 mM Tris HCl, 50 mM KCl, pH 8.4, 1.5 mMMgCl₂, and 2.5 U of Taq DNA polymerase (Life Science).

[0195] Generation of a EPR-1-specific antisense probe was carried out byaddition of 0.2 mg/ml of a “SacII” reverse oligonucleotide5′TGCTGGCCGCTCCTCCCTC3′, while extension of the EPR-1 positive strandand generation of a Survivin-specific probe was performed using 0.2mg/ml of forward F11 oligonucleotide 5′ATGACCTCCAGAGGTTTC3′. Twenty-fivecycles of amplification were carried with denaturation at 94° C. for 1min, annealing at 52° C. for 1 min, and extension at 72° C. for 1 min.The EPR-1 sense or antisense probes were centrifuged through a SephadexG-50 spin column (Worthington Biochemical Corp., Freehold, N.J.) at14,000 g for 5 min to separate free from incorporated radioactivity,heated at 100° C. for 2 min, and immediately added to the hybridizationreaction.

[0196] Identical strand-specific probes were used for hybridization ofmultiple tissue blots of adult or fetal human mRNA (Clontech), in5×SSPE, 10×Denhardt's solution, 2% SDS, 100 mg/ml denatured salmon spermDNA at 60° C. for 14 h, and washes at 60° C., as described above.Aliquots of total RNA extracted from undifferentiated or vitamin D₃terminally differentiated HL-60 cells, were processed for Northernhybridization with Survivin-specific single strand probe, as describedabove.

Example 2 Production of Anti-Survivin Antibodies

[0197] A Survivin sequence-specific antibody, called JC700, was producedand characterized as follows. A seventeenmer peptide corresponding tothe Survivin sequence A ³PTLPPAWQPFLKDHRI¹⁹ was synthesized andcharacterized by mass spectrometry. One hundred mg of the Survivinpeptide were coupled in a 1:1 ratio to Keyhole Limpet Hemocyanin andinjected s. c. into a rabbit in complete Freund's adjuvant. After a4-week interval, animals were boosted with s. c. injection of 100 mg ofpeptide in incomplete Freund's adjuvant and sequentially boosted andbled at alternate weeks.

[0198] Purification of the anti-Survivin antibody was carried out byaffinity chromatography on a peptide-Sepharose matrix (5 mg/ml ofpeptide) with elution of the specific IgG fraction in 1 mM glycine, pH2.5. Specificity of the affinity-purified anti-Survivin antibody,designated JC700, was determined by ELISA against the immobilizedSurvivin peptide or a control EPR-1 peptide by absorbance at OD₄₀₅.

Example 3 Production of a Monoclonal Antibody Against a Survivin FusionProtein

[0199] The Survivin cDNA was expressed as a GST-fusion protein in E.ColiBL21 strain and purified to homogeneity with removal of the GST frame.The purified protein was used to inject mice and generate monoclonalantibodies using standard hybridoma technology. Three independent mAbswere isolated, cloned twice by limiting dilution and furthercharacterized. One of the new anti-Survivin mAbs, designated 8E2,recognized the immobilized, purified recombinant Survivin by ELISA andbound to Survivin in immunoblots, as shown in FIG. 11.

Example 4 Immunoblotting and In Situ Hybridization

[0200] For immunoblotting, aliquots of SDS-solubilized extracts ofvarious transformed cell lines, non-transformed HUVEC, PBMC or Lu18, orundifferentiated or vitamin D₃-differentiated HL-60 cells, werenormalized for protein content by absorbance at OD₂₈₀, separated byelectrophoresis on a 5-20% SDS polyacrylamide gradient gel under nonreducing conditions, and electroblotted to Immobilon membranes(Millipore Corp., New Bedford, Mass.) at 1.1 A for 30 min at 22° C. Themembrane was blocked in TBS, pH 7.4, plus 5% milk, and incubated with 20mg/ml of control non-immune rabbit IgG or anti-Survivin antibody JC700for 1 h at 22° C., followed by washes in TBS, pH 7.4, and addition of a1:7500 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG(Promega) for 30 min at 22° C. Binding of the primary antibody wasrevealed by addition of 75 mg/ml nitro blue tetrazolium in 70%dimethylformamide (Sigma Chemical Co., St. Louis, Mo.) plus 50 mg/ml5-bromo-4chloro-3-indolyl phosphate (Sigma) in 100% dimethylformamide.

[0201] Tissue samples, immunohistochemistry and in situ hybridization.Tissue samples from colon adenocarcinoma (6 cases), lung squamous cellcarcinoma (6 cases), lung adenocarcinoma (9 cases), pancreasadenocarcinoma (2 cases), invasive breast adenocarcinoma (7 cases), wereobtained from the archives of Yale-New Haven Hospital and used in thepresent study. Samples of 44 high grade lymphoma tissues and 7 low gradelymphoma tissue was also obtained. Tissue samples were fixed informalin, embedded in paraffin, cut in 5 μm sections, deparaffinized inxylene, and rehydrated in graded alcohol followed by quenching ofendogenous peroxidase activity by treatment with 2% H₂O₂ in methanol.

[0202] For immunostaining, the slides were boiled for 5 min in astandard pressure cooker, blocked in 10% normal goat serum, andincubated with affinity-purified anti-Survivin antibody JC700 (20 μg/ml)for 14 h at 4° C. After washes in PBS, pH 7.4, slides were furtherincubated with biotin-conjugated goat anti-rabbit IgG (VectorLaboratories, Burlingame, Calif.) for 30 min at 22° C., and washed inPBS, pH 7.4. After addition of streptavidin-biotin conjugated peroxidase(Boehringer Mannheim) for 30 min at 22° C., slides were washed, andbinding of the primary mAbs was revealed by addition of3′-3′-diamino-benzidine (DAB) and counterstaining with hematoxylin.

[0203] Negative controls were carried out by replacing the primaryantibody with normal goat serum, under the same experimental conditions.In some experiments, aliquots of JC700 antibody were pre-adsorbed with25 mg/ml of the Survivin 3-19 peptide before tissue staining. For insitu hybridization, 1 μg of the Survivin cDNA containing the entirecoding sequence plus 271 bp of 3′ untranslated region in pcDNA3(Invitrogen), was completely digested with EcoRI and transcribed in theantisense orientation using T7 RNA polymerase in the presence ofdigoxigenin 11-uridine-5′ triphosphate (Boehringer Mannheim). Tissueslides were coated with 1% gelatin, 0.1% chrome-alum, baked at 120° C.for 2 h, and stored dust-free at 22° C. Sections were deparaffinized andrehydrated through graded alcohol, digested with proteinase K (1 μg/mlin 100 mM Tris HCl pH 8.7, 50 mM EDTA) for 30 min at 37° C., andacetylated in 0.25% acetic anhydride acid and 100 mM triethanolamine pH8.0 for 10 min at 22° C.

[0204] Detection of Survivin mRNA in human tissues was carried out by insitu hybridization of the Survivin antisense riboprobe in a buffercontaining 4×SSC, 1×Denhardt's solution, 50% deionized formamide, 250μg/ml yeast tRNA, 500 μg/ml salmon sperm DNA and 5% dextran for 16 h at50° C. After washes in 2×SSC for 90 min at 48° C., immobilizeddigoxigenin was detected using an anti-digoxigenin mAb (BoehringerMannheim) at a 1:3000 dilution, and revealed by alkaline phosphatasestaining with NBT/BCIP cytochemical stain.

Example 5 Expression of Survivin in Human Cancers

[0205] Survivin is prominently expressed in human cancer. For itsabundant distribution in transformed cell types, a potential expressionof Survivin in neoplasia was investigated, in vivo. Immunohistochemicalanalysis of formalin-fixed, paraffin embedded tissue sections with theaffinity-purified anti-Survivin JC700 antibody demonstrated abundantexpression of Survivin in all cases examined of human lung cancer,including adenocarcinoma (FIG. 6A), and squamous cell carcinoma (FIG.6C). Consistent with the topography of other IAP proteins (Duckett, C.S. et al., EMBO J (1996) 15:2685-2694), expression of the protein wasexclusively localized to the cytoplasm of tumor cells, while theadjacent normal gland epithelium of the lung did not express Survivin(FIG. 6C, arrow). No staining was observed when the anti-Survivinantibody was substituted with control goat serum (not shown), or afterpre-adsorption with the immunizing Survivin 3-19 peptide (FIG. 6B), thusconfirming the specificity of the observed recognition.

[0206] Prominent accumulation of Survivin mRNA in squamous lung cellcarcinoma was independently demonstrated by in situ hybridization with aSurvivin-specific single strand riboprobe (FIG. 6D). Survivin was alsoabundantly detected in all cases examined of adenocarcinoma of pancreas(FIG. 6E), and breast (not shown) by immunohistochemistry, and colon(FIG. 6G) by in situ hybridization. However, consistent with its absencein non-transformed cell types HUVEC and Lu18 (FIG. 4C), in maturetissues (FIG. 3), and in terminally-differentiated HL-60 cells (FIG. 5),no reactivity of the anti-Survivin JC700 antibody was observed withnormal exocrine pancreatic epithelial cells by immunohistochemistry(FIG. 6F), and no Survivin mRNA was found in the adjacent non-neoplasticcolon gland epithelium by in situ hybridization (FIG. 6H).

[0207] Expression of Survivin in Lymphoma Tissue. Tissue samples wereobtained from 44 patients with aggressive, high grade lymphoma and 7samples were obtained from 7 patients with non-aggressive, low gradelymphoma. The sample were treated as described above and examined forSurvivin expression. None of the low grade lymphoma samples displayedSurvivin expression whereas 27 samples (61%) from patients with highgrade lymphoma expressed Survivin.

Example 6 Expression of Survivin in other Cancers

[0208] In addition to the malignant forms of cancer discussed above, theexpression of Survivin in other types of cancers was investigated in theinventors' laboratory or collaboratively with other academicinvestigators. Survivin was found prominently expressed in the mostaggressive and metastatic forms of malignant thymoma (−100 casestested), in head and-neck squamous cell carcinoma (−140 cases) and inall forms of prostate cancer (15 cases), including the transition lesionof benign prostate hyperplasia. The most aggressive forms ofneuroblastoma are also positive for Survivin as discussed below.

Example 7 Tissue Specific Expression of Survivin

[0209] Survivin, was recently found in all the most common human cancersbut not in normal, terminally differentiated adult tissues. Theexpression of Survivin in embryonic and fetal development wasinvestigated. Immunohistochemistry and in situ hybridization studiesdemonstrated strong expression of Survivin in severalapoptosis-regulated fetal tissues, including the stem cell layer ofstratified epithelia, endocrine pancreas and thymic medulla, with apattern non-overlapping with that of another apoptosis inhibitor, i.e.bcd-2. A sequence-specific antibody to Survivin immunoblotted a single−16.5 kD Survivin band in human fetal lung, liver, heart, kidney andgastrointestinal tract. In mouse embryo, prominent and nearly ubiquitousdistribution of Survivin was found at embryonic date (E) 11.5, whereasat E15-21, Survivin expression was restricted to the distal bronchiolarepithelium of the lung and neural crest-derived cells, including dorsalroot ganglion neurons, hypophysis and the chorioid plexus. These datasuggest that expression of Survivin in embryonic and fetal developmentmay contribute to tissue homeostasis and differentiation independentlyof bcl-2.

Example 8 Preparation of Survivin Transfectants

[0210] Inducible Survivin antisense transfectants andapoptosis/proliferation experiments. A 708 bp SmaI-EcoRI fragmentcomprising nucleotides 379-1087 of the EPR-1 cDNA, was directionallycloned in the sense orientation in the mammalian cell expression vectorpML1 (generously provided by Dr. R. Pytela, Cardiovascular ResearchInstitute, University of California, San Francisco). The vector isderived from the episomal mammalian cell expression vector pCEP4 byreplacing the cytomegalovirus promoter cassette with the mMT1 promoter,directing Zn²⁺-dependent expression of recombinant proteins in mammaliancells (Lukashev, M. E. et al., J Bol Chem (1994) 269:18311-18314).

[0211] Ten million HeLa cells were incubated with 10 mg of pML1 DNAcontaining the Survivin antisense construct plus 50 mg of salmon spermDNA for 15 min on ice, followed by a single electric pulse delivered bya Gene Pulser apparatus (Bio-Rad) at 350 V at 960 μF. Forty-eight hafter transfection, cells were diluted fifteen fold, plated onto 100 mmdiameter tissue culture dishes and selected for 4 weeks in completegrowth medium containing 0.4 mg/ml hygromycin. Apoptosis in controlcultures or Survivin antisense HeLa cell transfectants was evaluated byin situ detection of internucleosomal DNA degradation afterZn²⁺-dependent induction of EPR-1 transcription under serum-starvingconditions.

[0212] Briefly, control or antisense Survivin transfectants were treatedwith 200 mM ZnSO₄ in 0% FBS for 24 h at 37° C. Cells were harvested,centrifuged at 800 g for 10 min at 4° C., and the pellet was fixed in10% formalin overnight, dehydrated, embedded in paraffin blocks, andsections of 3-5 mm were put on high adhesive slides. Samples weretreated with 20 mg/ml proteinase K for 15 min at 22° C., washed indistilled water, quenched of endogenous peroxidase in 2% H₂O₂ in PBS,and subsequently mixed with digoxigenin-labeled dUTP in the presence ofterminal deoxynucleotidyl transferase (TdT) followed by peroxidaseconjugated anti-digoxigenin antibody.

[0213] Nuclear staining in apoptotic cells was detected by DAB,according to the manufacturer's instructions (AptoTag, Oncor,Gaithersburg, Md.). Control experiments were performed by omitting theenzyme incubation step. Morphologic features of apoptotic cells(apoptotic bodies) under the various conditions tested were detected byhematoxylin/eosin staining of the same slides.

[0214] For proliferation experiments, vector control HeLa cells orSurvivin antisense transfectants were plated at 20×10⁴/well onto 24-welltissue culture plates (Costar), induced with 200 mM ZnSO₄ for 16 h at37° C., harvested at 24 h intervals, and cell proliferation under thevarious conditions tested was determined microscopically by direct cellcount. Down-regulation of Survivin expression under these experimentalconditions was assessed by immunoblotting with JC700 antibody.

Example9 Identification of EPR-1 Complementary Gene

[0215] Three overlapping clones were isolated by hybridization screeningof a human P1 plasmid genomic library with the EPR-1 cDNA and confirmedby Southern blot. This gene was located to the long arm of chromosome17, to band 17q25, by fluorescence in situ hybridization (FIG. 1A, B).

[0216] A contig of P1 fragments spanning 14796 bp was cloned in pBSKS⁻and completely sequenced on both strands (FIG. 1C). Three putativesplice sites, matching perfectly the consensus sequences for eukaryoticintron-exon boundaries (Padgett, R. A. et al., Ann Rev Biochem (1986)55:1119-1150), were identified at position 2922, 3284, and 5276 (donor),and 3173, 5157, and 11954 (acceptor), thus defining a gene organizationin four exons and three introns of 252, 1874, and 6678 bp, respectively(FIG. 1D).

[0217] Sequence analysis of the putative coding regions demonstrated anearly complete identity with the EPR-1 cDNA (Altieri, D. C., FASEB J(1995) 9:860-865), except for 5 nucleotide changes and 6 nucleotideinsertions. However, the three splice sites were found on thecomplementary, antisense strand of the EPR-1 coding sequence. Consistentwith this unexpected orientation, the EPR-1 complementary gene revealeda 5′ GC rich region, comprising nucleotides 2560-2920 and including exon1 (see below), which fulfilled the base composition criteria of a CpGisland (Gardiner-Garde, M. et al., J Mol Biol (1987) 196:261-282 andFrommer, 1987). Sequencing the 2.5 kb upstream the CpG island revealed aTATA-less promoter with numerous Sp1 sites (not shown).

[0218] Complex hybridization pattern and evolutionary conservation ofEPR-1 sequences. Probing human genomic DNA with the EPR-1 cDNA revealedseveral hybridizing fragments (FIG. 2A). Of these, a ˜7.5 kb XbaI, a 7.6kb BamHI, and 4 HindIII fragments of ˜15, 7.5, 6.4, and 3.7 kb,respectively (FIG. 2A, arrows), could not be recapitulated by therestriction map of the antisense EPR-1 gene (FIG. 1C). In contrast,other bands of comparable intensity, including a 5.15 kb XbaI and a 7.1kb BamHI fragment, genuinely originated from the antisense EPR-1 geneand comprised the first two, or three exons, respectively (FIG. 2A).

[0219] At variance with this complex hybridization pattern, Southernblot of high molecular weight human genomic DNA digested with MluI orNotI and separated by pulsed field gel electrophoresis, revealed singleEPR-1-hybridizing bands of ˜75 kb and 130 kb, respectively (FIG. 2B).Finally, Southern blots of multiple species genomic DNA revealedsignificant evolutionary conservation of EPR-1-related sequences (FIG.2C), with numerous strongly hybridizing bands in mammalian species andfainter signals in rabbit or chicken genomic DNA, under high stringencyhybridization conditions (FIG. 2C).

[0220] Discordant tissue distribution of sense/antisense EPR-1transcripts. The potential expression of distinct sense or antisenseEPR-1 transcripts was investigated in Northern blots with singlestrand-specific probes. Consistent with the size of the spliced EPR-1message (Altieri, D. C., FASEB J (1995) 9:860-865), an EPR-1strand-specific probe detected a prominent ˜1.2 kb band in mRNAextracted from all adult and terminally-differentiated human tissuesexamined (FIG. 3A). In contrast, no specific bands hybridized with aEPR-1 antisense-specific single strand probe in adult tissues, under thesame experimental conditions (FIG. 3B). A similar ˜1.2 kb band wasdetected by the single strand EPR-1-specific probe in fetal kidney, and,to a lesser extent, in fetal liver, lung and brain (FIG. 3A). Atvariance with the absence of hybridization in adult tissues, the EPR-1antisense-specific probe recognized a prominent ˜1.9 kb band, and alarger 3.2 kb species corresponding to the size of an incompletelyprocessed transcript, in fetal liver, while fainter hybridization bandswere also seen in fetal kidney, lung and brain (FIG. 3B). A controlhybridization with an actin probe confirmed comparable loading of mRNAin adult or fetal samples (FIG. 3C).

[0221] Characterization of the antisense EPR-1 gene product. Inspectionof the 5′ CpG island in the antisense EPR-1 gene revealed a putative ATGinitiation codon at position 2811, surrounded by a sequence (CGGCATGG)that conformed well to the consensus for eukaryotic initiation oftranslation (Kozak, M., Nucleic Acids Res (1984) 12:857-872). Analysisof the antisense EPR-1 sequence in the 5′→3′ direction dictated by theposition of intron-exon boundaries revealed an open reading frame of 426bp, spanning all four exons, and terminating with a TGA codon atposition 12042 in exon 4. A canonical polyadenylation signal (AATAAA)was found at position 13166. PCR products amplified fromreverse-transcribed HeLa cell RNA primed with EPR-1 “sense”oligonucleotides matched perfectly the genomic sequence and confirmedthe open reading frame and the predicted intron-exon boundaries (notshown).

[0222] Two λgt11 cDNA clones isolated by hybridization of a HEL librarywith the EPR-1 CDNA, also matched the consensus genomic sequence andrevealed a homopolymeric A tail on the antisense EPR-1 strand atposition 13186, 14 bp downstream the polyadenylation signal, generatinga 3′ untranslated region of 1144 bp. In these clones, the 5′untranslated region upstream from the initiating ATG was of 49 bp,beginning at position 2762 in the genomic sequence, and contained anin-frame termination codon. Translation of the antisense EPR-1 openreading Same predicted a new protein of 142 amino acids, with anestimated molecular weight of 16,389 and an acidic pI of 5.74, lackingan amino-terminus signal peptide or a carboxy terminus hydrophobicstretch for membrane insertion (FIG. 4A).

[0223] A coiled coil was predicted for the last 40 carboxy terminusresidues (Lupas, A. et al., Science (1991) 252:1162-1164). BLASTdatabase searches revealed a significant degree of similarity betweenresidues 18-88 of the antisense EPR-1 gene product and the BIR module inthe IAP family of inhibitors of apoptosis (Birnbaum, M. J. et al., JVirology (1994) 68:2521-2528; Clem, R. J. et al., Mol Cell Biol (1994)14:5212-5222). For this analogy, the antisense EPR-1 gene product wasdesignated Survivin. At variance with other IAP proteins, Survivincontained only one BIR, encoded by the first three exons of the gene,and lacked a carboxy terminus RING finger, without additional/alterativeexon(s) potentially encoding this domain (FIG. 1C).

[0224] An alignment by the Clustal method between the Survivin BIR andthat of other known IAP proteins is shown in FIG. 4B. Despite theoverall match of the consensus and several conservative substitutions,phylogenetic analysis suggested that Survivin is a distantly relatedmember of the IAP family, most closely related to NAIP, which alsolacked a RING finger (FIG. 4B, shaded boxes) (Roy, N. et al., Cell(1995) 80:167-178).

[0225] A rabbit polyclonal antiserum designated JC700, was raisedagainst residues A³PTLPPAWQPFLKDHRI¹⁹ of Survivin, purified by affinitychromatography on a peptide-Sepharose column, and used in Western blots.Consistent with the predicted molecular weight of Survivin, JC700antibody immunoblotted a single band of ˜16.5 kDa fromdetergent-solubilized extracts of all transformed cell lines examined,including B lymphoma Daudi and JY, T leukemia Jurkat and MOLT13,monocytic THP-1, and erythroleukemia HEL (FIG. 4C).

[0226] Survivin was also found in isolated peripheral blood mononuclearcells (PBMC). In contrast, no expression of Survivin was detected innon-transformed Lu-18 human lung fibroblasts or human umbilical veinendothelial cells (HUVEC) (FIG. 4C). No specific bands wereimmunoblotted by control non-immune rabbit IgG, under the sameexperimental conditions (FIG. 4C).

[0227] Identification of agents that modulate transcription of the EPR-1gene. Agents that increase the transcription of the EPR-1 gene may beidentified by conventional techniques. Preferably, a candidate agent isbrought into contact with a cell that expresses the EPR-1 gene productand the level of expression of this product or the level oftranscription are determined and agents that increase or decrease EPR-1gene transcripts may readily be identified. Alternatively, the EPR-1transcriptional regulatory elements may be placed upstream of a reportergene such as CAT or β-galactosidase.

Example 10 Regulation of Survivin Expression by CellGrowth/Differentiation

[0228] Consistent with the expression of Survivin in transformed celllines (FIG. 4C), undifferentiated and actively proliferatingpromyelocytic HL-60 cells constitutively expressed high levels ofSurvivin, as demonstrated by immunoblotting of a single ˜16.5 kDa bandwith JC700 antibody, and Northern hybridization of a ˜1.9 kb transcriptwith a single strand-specific probe (FIG. 5). In contrast, no specificbands were recognized by control non-immune rabbit IgG under the sameexperimental conditions (FIG. 5).

[0229] Vitamin D₃-induced terminal differentiation of HL-60 cells to amature monocytic phenotype resulted in growth arrest of these cells andde novo induction of differentiation-specific markers, including a˜200-fold increased expression of leukocyte CD11b/CD18 integrin detectedby flow cytometry (not shown), and in agreement with previousobservations (Hickstein, D. D. et al., J Immunol (1987) 138:513-519).Under these experimental conditions, the anti-Survivin JC700 antibodyfailed to immunoblot specific bands from vitamin D₃-treated HL-60extracts, and no Survivin transcript(s) were detected by Northernhybridization with a single strand-specific probe (FIG. 5).

[0230] In contrast, an anti-EPR-1 polyclonal antibody immunoblotted asingle ˜62 kDa band corresponding to EPR-1 in vitamin D₃-differentiatedHL-60 extracts under the same experimental conditions (not shown).Moreover, down-regulation of Survivin in vitamin D₃-differentiated HL-60cells was accompanied by a 5- to 10-fold increased surface expression ofEPR-1 in these cells, as detected by flow cytometry with anti-EPR-1monoclonal antibodies B6 or 12H1 (FIG. 8).

[0231] As shown in FIG. 16, Survivin is down regulated by thecombination of cytokines γ interferon and tumor necrosis factor α, butnot by either cytokine alone. Similarly, the transfection of 3T3 cellswith the c-myc oncogene results in the up-regulation of Survivin mRNA asdetected by Northern blots.

Example 11 Promoting Apoptosis with Survivin

[0232] Targeting Survivin promotes apoptosis and inhibits cellproliferation. Transfection of the Survivin cDNA in mouse or hamstercell lines (NIH 3T3, CHO) was not suitable for the presence ofimmunochemically indistinguishable endogenous homologues in these cells(not shown). Similarly, initial attempts to target the Survivin gene instable antisense transfectants were unsuccessful for slow cell growthand rapid loss of viability (not shown). Therefore, Survivin⁺ HeLa cellswere transfected with the 3′ end of the EPR-1 cDNA (Survivin antisense)under the control of a metallothionein-inducible promoter (Lukashev, M.E. et al., J Biol Chem (1994) 269:18311-18314), selected in hygromycin,and analyzed for apoptosis and cell proliferation after Zn²⁺-dependentactivation of transcription.

[0233] Consistent with the expression of Survivin in transformed celllines (FIG. 4C), the JC700 antibody immunoblotted a single molecularspecies of ˜16.5 kDa in extracts of control HeLa cells transfected withthe vector alone (FIG. 7A). In contrast, no specific bands wererecognized by JC700 antibody in metallothionein-induced HeLa cellstransfected with the EPR-1 cDNA (Survivin antisense) (FIG. 7A). Underthese experimental conditions, in situ analysis of internucleosomal DNAfragmentation by AptoTag staining revealed only a few apoptotic cells inserum-starved, Zn²⁺-induced, vector control HeLa cells (FIG. 7B).

[0234] In contrast, as discussed above, inhibition of Survivinexpression in Zn²⁺-induced antisense HeLa cell transfectants wasassociated with prominent nuclear staining in the vast majority of cellsexamined (FIG. 7B). No nuclear staining was detected in the absence ofTdT tagging of the digoxigenin-labeled dUTP (not shown). Typicalmorphologic features of apoptosis, including numerous apoptotic bodies,were also demonstrated in induced antisense HeLa cell transfectants byhematoxylin/eosin staining, while only occasional apoptotic bodies wereobserved in vector control HeLa cultures, under the same experimentalconditions (FIG. 7B).

[0235] A potential effect of Survivin on cell growth was alsoinvestigated. In these experiments, metallothionein-controlled,EPR-1-dependent, inhibition of Survivin expression caused a profoundreduction of serum-dependent HeLA cell proliferation (FIG. 7C). Threedays after Zn²⁺ induction, the cell count in vector control HeLacultures increased by 288%, as opposed to only a 20% increase inSurvivin antisense transfectants, under the same experimental conditions(FIG. 7C).

Example 12 Structure—Function Relationship of Survivin

[0236] The minimal structural requirements involved in Survivin-mediatedinhibition of apoptosis have been identified through a mutagenesisstrategy of Ala substitutions of the most evolutionarily conservedresidues in the single Survivin BIR (baculovirus IAP repeat) module.These residues included in the amino-terminal half of the Survivin BIR,Arg¹⁸, Phe²², Trp²⁵, Pro²⁶, Pro³⁵, Ala³⁹, Ala⁴¹, Gly⁴², and Cys⁴⁶. Inthe carboxyl-terminal half of the Survivin BIR, Ala mutants were firsttargeted at the CyS⁵⁷X₂Cys⁶⁰X₁₆His⁷⁷X₆Cys⁸⁴ putative zinc binding motif.Additional conserved residues targeted by mutagenesis include Asp⁵³ ,Leu⁶⁴, Trp⁶⁷, Pro⁶⁹, Asp⁷¹, Asp⁷² and Pro⁷³.

[0237] The Survivin mutants are characterized in stable and transientlytransfected cells, IL-3-dependent BaF3 cells and NIH3T3, respectively.In addition to these point mutants, a Survivin chimeric moleculecontaining a carboxyl-terminal RING finger has also been generated andscreened for apoptosis inhibition (the RING finger is a domain found inmost other IAP proteins, but not in Survivin). Secondly, a truncatedform of Survivin has also been generated, in which the last 40carboxylterminus residues, containing a predicted coiled-coil structure,have been deleted. As shown in FIG. 12, Ala mutagenesis of key conservedresidues in Survivin Trp⁶⁷-Pro⁷³-Cys⁸⁴ produced a recombinant moleculewhich lacked the ability to protect BaF3 cells from apoptosis induced byIL-3 withdrawal.

Example 13 Cytoprotective Effects of Survivin

[0238] Classical examples of cell damage to stable cell populationsmediated by apoptosis include allograft rejection by infiltratinglymphocytes, Alzheimer's disease and reperfusion injury followingmyocardial infarction. In addition to being expressed in cancer, therebyfunctioning as a growth-advantage factor for cancer cells, the targetedexpression of Survivin is useful to protect stable cell populations fromapoptosis and other cellular insults. This application of Survivin wastested by adding increasing concentrations of purified recombinantSurvivin to monolayers of human endothelial cells injured with hydrogenperoxide (H₂O₂), a classical apoptosis-inducing stimulus. The resultsare summarized in FIG. 13. Increasing concentrations of added Survivinresulted in a significant increased viability of the treated cells asopposed to control cultures treated with control protein myoglobin.Similarly, Survivin protected NIH3T3 cells from apoptosis induced byhydrogen peroxide after transient co-transfection with a lacZ reportergene as shown in FIG. 17.

Example 14 Survivin as a Predictive-Prognostic Factor

[0239] The presence of Survivin can be utilized as predictive-prognosticnegative factor in neuroblastoma and non-Hodgkin's lymphoma, and inother cancers.

[0240] Neuroblastoma. A large series of neuroblastoma cases (72) wasscreened for Survivin expression in a multicentric study. As shown inFIG. 14, Survivin expression increased dramatically when patientscontained at least one negative prognostic factor for aggressive andrapidly progressing disease. Secondly, expression of Survivin stronglycorrelated with a more aggressive disease and unfavorable histology.Importantly, expression of Survivin was a more sensitive prognosticindex than simple histology. Survivin-positive cases with earlydiagnosis of favorable histology were found to contain at least onenegative prognostic factor for disease progression and dissemination.

[0241] Hodgkin's Lymphoma. A similar multicentric study has beenrecently completed on analysis of Survivin expression in high gradenon-Hodgkin's lymphoma (n=8). The results are similar to those observedfor neuroblastoma. As shown in FIG. 15, expression of Survivin stronglycorrelated with a more widespread disease predominantly in stage IV.Clinically, Survivin-expressing patients had fewer episodes of completeremission and more episodes of incomplete remission, no remission orrelapses as compared with Survivin-negative patients.

[0242] Potential implications. The demonstrated role of Survivin as anegative predictive prognostic factor in these two embryologicallydifferent types of cancer iterates the potential use of this molecules adinnostic tool to monitor disease progression and response to thetherapy. It can also be used for staging purposes and to identifypopulations of patients potentially susceptible to multi-drug resistance(groups with no remissions or incomplete remissions). Also, Survivinderived primers easily designed from the complete sequence of theSurvivin gene can be used as a screening tool to identify potentialcases of cancer in which the Survivin gene has been deleted or mutated.These cases will be very important to identify because targetedinactivation of the Survivin gene would confer a favorable prognosticfactor to cancer patients, removing a potential drug-resistance gene.Inactivating mutations in the Survivin gene can target the same keyresidues identified in our initial screening of Ala-based mutagenesis orresult in an abortive or truncated protein for premature termination oftranslation.

Example 15 Survivin Cancer Vaccine

[0243] Vaccines directed against Survivin, as found in various types ofcancer, may be developed as with other disease-related intracellularprotein targets. These techniques are commonly available andrepresentative approaches are described by the references cited below.Vaccines may also include the systemic administration of peptidefragments of Survivin and the use of vectors to deliver mini-genesencoding Survivin peptides to tumor cell targets are contemplated. Asmentioned above, Survivin is not expressed in normal cells, even inproliferating stem cells in the bone marrow. This ensures that theimmune response mounted against Survivin will be highly selective andspecific and will not involve normal cells.

[0244] Development and Administration of Polypeptide-based vaccines

[0245] Methods of rthe use of peptide components in a monovalent or apolyvalent cancer immunotherapy-vaccine product are described by Nardi,N. et al., Mol. Med. (1995) 1(5):563-567. Additional references thatdiscuss the different cancer vaccine and cancer immunotherapiescurrently being used include: N. P. Restifo and M. Sznol “CancerVaccines,” in DeVita's Cancer: Principles & Practice of Oncology3023-3043 (Lippincott-Raven, Philadelphia; 1997); J. Galea-Lauri et al.,Cancer Gene Ther. (1996) 3(3): 202-214; D. C. Linehan et al., Ann. Surg.Oncol. (1996) 3(2): 219-228; and J. Vieweg et al., Cancer Invest. (1995)13(2): 193-201.

[0246] Consistent with the foregoing approach, Survivin polypeptides orfull length Survivin are synthesized either chemically by knowntechniques or recombinantly by expressing appropriate cDNAs inprokaryotic or eukaryotic cells. Survivin proteins so produced are thenpurified as necessary to remove contaminating proteins, such as serum orbacterial proteins. Survivin can be further purified using columnscontaining antibodies that bind Survivin, such as the monoclonalantibody JC700 or the antibody 8E2 (both described above) whichrecognize and bind to Survivin. In purifying an antibody-based vaccine,the recombinantly produced Survivin would bind to the antibodies whileother proteins and cellular debris would be washed out. Survivinpolypeptides are then be isolated and concentrated to a desiredstrength.

[0247] Alternatively Survivin polypeptides are created by cleaving thenative Survivin with one or more proteases (e.g., trypsin). Proteolyticfragments are then be separated and recovered using SDS-PAGE,high-resolution/high-pressure separation techniques, or reverse-phaseHPLC. See R. J. BEYNON AND J. S. BOND, PROTEOLYTIC ENZYMES: A PRACTICALAPPROACH (Oxford University Press, New York 1989). These isolatedpeptides are then be concentrated to a desired final concentration.

[0248] Once purified, Survivin polypeptides or full length Survivinmolecules may then placed in an emulsion containing an adjuvant.Adjuvants contemplated for use with Survivin include aluminum adjuvants,Freund's adjuvant, oil-in-water emulsions containing tubercle bacilli,and interleukin-2 (IL-2). Additional preparations include combining theSurvivin polypeptides with other appropriate tumor-associated antigensand, optionally, other immunomodulatory agents such as cytokines. Othersuitable carriers or excipients can be used including bovine serumalbumin, coupling the Survivin polypeptide with haptens, keyhole limpethemocyanin, ovalbumin, and purified protein derivative of tuberculin.Peptides may be coupled to carriers using techniques such as thosedescribed in ED HARLOW AND DAVID LANE, ANTIBODIES: A LABORATORY MANUAL(Cold Spring Harbor Laboratory, 1988).

[0249] Vaccines in human subjects may be administered in the form of anemulsion injected subcutaneously, intradermally or intramuscularly (IM);vaccines appropriately formulated can be taken orally. With vaccinescontaining adjuvants, the vaccine is generally preferably be given IM,e.g., in the deltoid.

[0250] The amount of Survivin vaccine or Survivin peptide vaccine to beadministered to a patient will correspond to values typically used infor other cancer vaccines. Dosage concentrations will range from about0.25 g to about 1000 g per day. More preferred ranges will be from about10 μg to about 500 μg per day.

Example 16 Diagnostic use of Anti-Survivin Antibodies

[0251] Frequently, tumor associated antigens (TAA) are shed from tumorcells into the surrounding plasma or into the blood. As a result, TAAoften are found in the blood, and blood samples obtained from patientsmay be used in detecting the presence of cancer, as well as used as afactor is staging cancers (e.g., stage I, II, III or IV). Survivin isone such TAA, and healthy, normal individuals do not express Survivin.Results from studies of several cancers have indicated that the presenceof Survivin (or Survivin fragments) correlates with and is predictivethat the disease may be aggressive or may have metastasized. A similarstrategy of detecting and quantifying the levels of Survivin or Survivinfragments can be used to determine residual tumor burden in patientsundergoing chemotherapy or radiation therapy for cancer treatment.Elevated or increasing levels of Survivin may reflect late stageneoplastic disease.

[0252] For diagnostic uses, blood is drawn from patients, by well knowntechniques, who have known cancer loads or from patients suspected ofhaving cancer. The blood sample is prepared by known techniques and istested for binding with antibodies to Survivin that are prepared and,optionally, labeled, as discussed above. Such general antibody detectionprotocols and associated reagents are well established in the art. Otherbiological fluid samples such as semen, urine, or saliva can also bemonitored for the presence of Survivin. This diagnostic technique asocan be used to monitor disease progression and response toindividualized therapies. This method offers a relatively non-invasivemeans of tracking cancer progression or regression.

Example 17 Detection of Survivin by Immunobioassay

[0253] An illustrative example of an immunobioassay to test for thepresence of Survivin in the blood of patient relies on the ability ofthe monoclonal antibodies to Survivin to bind Survivin and remove thedetectable Survivin from solution by immunoprecipitation. Such animmunobioassay is used to detect Survivin in suspected cancer patientsand in fractions eluted from fractionation columns. An aliquot of eachpatient sample is incubated for 2 hours at 4° C. with a monoclonalantibody that specifically recognizes and binds Survivin, such as theMab 8E2, described above. The monoclonal antibody is insolubilized onanti-mouse IgG agarose beads, which can be acquired from Sigma ChemicalCo., St. Louis, Mo.

[0254] The agarose bead anti-mouse (IgG(H+L))-Survivin complex isprepared by first washing the agarose beads with binding buffercontaining 0.01 M phosphate buffer, (pH 7.2), and 0.25 M NaCl and thenincubating the beads with the Survivin monoclonal antibody for 18 hoursat 4° C. in the same buffer. The agarose beads may then be sedimented bycentrifugation for 30 seconds at 16,000×g in a microcentrifuge andnon-specific sites may be blocked by incubation with 2% non-fat dry milkin 0.5 M NaCl-TMK for 30 minutes at 4° C. After blocking, the beads maybe washed 3 times with 0.5 M NaCl-TMK and resuspended in an equal volumeof the same buffer. 20:1 of the agarose bead-monoclonal antibody complexmay then incubated with each 250:1 of the patient test sample for 2hours at 4° C. Any Survivin present in the patient test sample will befound by the Survivin monoclonal antibody on the beads. The beadcomplex, now with Survivin bound, may be removed by centrifugation for30 seconds at 16,000×g. The supernatant is then assayed for Survivinactivity in the bioassay as described below. Control samples are treatedwith blocked beads that lacked the Survivin monoclonal antibody andtested for Survivin activity in the bioassay.

Example 16 Detecting Survivin using a Direct Elisa Test

[0255] Samples of normal plasma (control) and cancer patient-plasma arediluted 1:1 with phosphate buffered saline (PBS). One volume of eachmixture is added to centricon-10 filter having a 10 kD molecular weightlimit and centrifuged at 5000×g (7000 rpm) for 1 hour. One volume of PBSis added to the retentate and centrifuged for 30 min. The final dilutionis about 1:3. The ELISA plate wells are then coated with retentate at1:6, 1:12, 1:24, 1:48 and 1:96 final dilution in bicarbonate coatingbuffer, having a pH 9.6 overnight at 4° C. C. The plates are then washed2 times with wash buffer containing 5% Tween 20 in phosphate bufferedsaline. Residual binding sites are blocked with 4% bovine serum albumin(BSA), 300 μ/well for 2 hours. Plates are then washed 2 times with washbuffer. Next, 100 μl of a monoclonal antibody that specificallyrecognizes and binds to Survivin, such as Mab 8E2, is used at 1:200dilution in 1% BSA is added to the wells and incubated for 1 hour withagitation. Plates are washed 5 times with wash buffer. Next, 100 μlhorseradish peroxidase conjugated secondary antibody is added, typicallyat a 1:2,000 dilution to each well, and incubated for 1 hour. Plates areagain washed 5 times with wash buffer. Next, 100 μl/well of substratecontaining 5 μg of Survivin and 5 μl H₂O₂/10 ml citrate-phosphate bufferis added to each well and incubated for 5 minutes. The enzyme reactionis stopped by adding 50 μl/well 2 M H₂SO₄. The absorbance of light ismeasured at 492 nm in an EIA reader. Patient samples that containSurvivin will produce a positive reading, whereas those samples that donot contain Survivin will be negative.

Example 18 Survivin Fragments, Peptides and Small Molecule Antagonists

[0256] As described above, key functional residues in Survivin requiredfor apoptosis have been identified. These data provide a template uponwhich to produce synthetic peptides and small molecule antagonists andcompetitive inhibitors of Survivin function. Preferably, the peptidesare produced from native Survivin or include substitutions from thenative Survivin peptide backbone that include the functionally relevantresidues Trp⁶⁷-Pro⁷³-Cys⁸⁴. Peptide fragments of native Survivin can begenerated by standard techniques, including protein digests. Adetermination of which fragments compete with Survivin can readily bemade by using the apoptosis measurement systems and apoptosis assaysystems described above. These results provide a unique opportunity toidentify a discrete linear sequence in Survivin, that is essential forinhibition of apoptosis.

[0257] Consistent with the general paradigm of IAP proteins-dependentinhibition of apoptosis, it also was predicted that a structural regionin the molecule required for the anti-apoptotic function is the primarycandidate for being a site of interaction with other molecules (such asbinding partners). The functionally relevant peptide sequence inSurvivin, based on the mutagenesis data, is: EGWEPDDDPIEEHKKHSSGC. Alasubstitutions of the underlined residues results in a complete loss offunction of Survivin in transfected cells. This linear sequence can besynthesized and used as a much more stringent and specific reagent toisolate associated molecules using standard biochemical procedures ofaffinity chromatography or as a bait for the yeast two-hybrid system.

[0258] Also, preferably, the βCOOH coiled-coil region of Survivin isincluded in Survivin fragments and peptides. Recent data indicates thatthis Survivin domain is important for the anti-apoptosis function ofSurvivin. We have generated a recombinant truncated form of Survivinlacking the last 40 βCOOH terminus amino acids comprising thecoiled-coil domain. This truncated form was co-transfected with a lacZplasmid in NIH3T3 cells side-by-side with wild type Survivin and XIAP,another member of the IAP gene family. The results, shown in FIG. 17,indicate that the truncated Survivin had lost most (˜80%) of thecytoprotective effect at preventing apoptosis in transfected cellsinduced by hydrogen peroxide. Incidentally, in this system, Survivin wasmore potent than NAIP at preventing apoptosis.

[0259] Agonists and antagonists of Survivin also can readily beidentified through conventional techniques. Designed, synthetic peptidesbased on the native linear sequence also function as competitiveinhibitors of Survivin's interaction with as yet unidentified partnermolecules. However, this inhibition should be sufficient to block theanti-apoptosis function of Survivin.

[0260] A similar peptide-based strategy has been successful to blockcaspase activation in vitro and in vivo, protecting cells fromapoptosis. See, e.g., Milligan, C. E. et al., (1995) Neuron 15:385-393.

Example 19 Therapeutic uses of Antisense Survivin DNA

[0261] As described above, the transcription of a Survivin antisensesequence altered the EPR-1/Survivin gene balance. This was demonstratedin HeLa cell transfectants, in which metallothionein-inducedtranscription of the EPR-1 “sense” strand suppressed the expression ofSurvivin and profoundly influenced apoptosis/cell proliferation.Additionally, transiently co-transfecting a Survivin antisense constructwith a lacZ reported plasmid decreased the viability of Survivinantisense trasfectants after a 48-h transfection in β-galactosidaseexpressing cells. Accordingly, the level of expression of Survivin in aSurvivin expressing cell or tissue, such as a tumor, is decreased bytrasfecting the cell or tissue with the EPR-1 sense strand of DNA.Alternatively, a Survivin antisense-encoding DNA is used to trasfect atarget cell or tissue. Such therapy effectively decreases thetranslation of Survivin-encoding mRNA into Survivin protein.

Example 20 Use of Survivin as a Protective Agent Against Apoptosis

[0262] Survivin has been shown to protect cells from apoptosis whenadministered to cells that have been exposed to hydrogen peroxide orother agents that typically induce apoptosis. It is contemplated thatcellular permeability may need to be increased, preferably in atransient manner in order to facilitate delivery of Survivin, orfragments thereof effective to reduce apoptosis. Certain conditionsinvolving transient metabolic inhibition or transient hypoxia are likelyto increase cellular permeability without the need for additional,external agents. Agents that may be appropriate include, metabolicinhibitors like 2-deoxygluocose and sodium azide, However, the abilityof Survivin to mediate cytoprotection during a transient increase incellular permeability offers the possibility of using therapeuticinfusion of recombinant Survivin to decrease reperfusion injury andcellular damage during myocardial infarction and stroke. It iscontemplated that such processes are mediated by increased tissue damagedue to apoptosis. Treatment with Survivin could reduce the extent andmagnitude of the injured tissue.

[0263] The use of Survivin or allelic varients of Survivin in subjectsto modulate or prevent apoptosis related cell death would be beneficialin treating or ameliorating the effects of a variety ofapoptosis-related indications. These indications include, but are notlimited to, dermatological effects of aging (e.g., baldness that iscaused by apoptosis of cells of hair follicle cells), disorders anddiseases such as immunosuppression, gastrointestinal perturbations(e.g., damage of lining of the gut, ulcers, and radiation orchemotherapy induced damage), cardiovascular disorders, apoptosisrelated to reperfusion damage (e.g., coronary artery obstruction,cerebral infarction, spinal/head trauma and concomitant severeparalysis, damage due to insults such as frostbite or burns, and anyindication previously thought to be treatable by superoxide dismutase),rejection of tissue transplantation (e.g., graft versus host disease),and Alzheimer's disease. The administration of Survivin also may becytoprotective against chemotherapy or radiation-induced apoptosis.

[0264] Survivin protein for administration can be produced as describedabove, e.g., using the cDNA described herein. The protein may requirepurification for purposes of pharmaceutical administration and suchpurification steps preferably utilize monoclonal antibody separation andpurification techniques as also described above.

[0265] In a clinical setting, Survivin is administered to patients inpharmaceutically effective dosages, i.e., in dosages effective to reducethe level or extent of apoptosis otherwise present, via several routes.For example, to treat dermatological ailments that involve apoptosis,Survivin can be administered in a salve, cream, ointment or powder form.Topical formulations may contain additional pharmaceutical or cosmeticcompositions such as moisturizers, humectants, odor modifiers, buffer,pigment, preservatives, vitamins (such as A, C or E), emulsifiers,dispersing agents, wetting agents, stabilizers, propellants,antimicrobial agents, sunscreen, enzymes and the like. Typical dosagesof Survivin that may be administered to patients will be 0.01% to 1.0%by weight. Additional topical pharmaceutical compositions are describedin S. Nakai et al., U.S. Pat. No. 5,672,603.

[0266] Survivin may also be administered, as may be appropriate for thecondition being treated, in the form of pills, solutions, suspensions,emulsions, granules or capsules. Survivin can be administered orally;injected in solutions administered intravenously either alone or inadmixture with conventional fluids for parenteral infusion (e.g., fluidscontaining glucose, amino acids etc.); injected intramuscularly,intradermally, subcutaneously or intraperitoneally; using suppositories;and in the form of ophthalmic solutions such as eye drops. Survivin canalso be administered using delayed release carriers, such as liposomes,microsponges, microspheres or microcapsules that are deposited in closeproximity to the tissue being treated for prevention of apoptosisrelated cell death.

[0267] Concentrations of Survivin or functional allelic variants ofSurvivin administered via routes other than topical administrationtypically would range in dose from about 10 μg per day to about 25 mgper day depending on the route of administration. Of course, it would beexpected that skilled artisans, such as physicians, may alter thesevalues on a case by case basis as required for the particular patient.

1 35 19 base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” 1 TGCTGGCCGC TCCTCCCTC 19 18 base pairs nucleic acidsingle linear other nucleic acid /desc = “oligonucleotide” 2 ATGACCTCCAGAGGTTTC 18 17 amino acids amino acid <Unknown> linear peptide 3 Ala ProThr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp His Ar 1 5 10 15 Ile 20amino acids amino acid <Unknown> linear peptide 4 Glu Gly Trp Glu ProAsp Asp Asp Pro Ile Glu Glu His Lys Lys Hi 1 5 10 15 Ser Ser Gly Cys 2027 base pairs nucleic acid single linear DNA (genomic) 5 GCGGGTGAGCTGTCCCTTGC AGATGGC 27 27 base pairs nucleic acid single linear DNA(genomic) 6 CCATGTAAGT TGATTTTTCT AGAGAGG 27 27 base pairs nucleic acidsingle linear DNA (genomic) 7 AATTGTATGT CTTTATTTCC AGGCAAA 27 45 aminoacids amino acid <Unknown> linear protein 8 Glu Glu Ala Arg Leu Val ThrPhe Gln Asn Trp Pro Asp Ala Phe Le 1 5 10 15 Thr Pro Gln Glu Leu Ala LysAla Gly Phe Tyr Tyr Leu Gly Arg Gl 20 25 30 Asp Gln Val Gln Cys Phe AlaCys Gly Gly Lys Leu Ala 35 40 45 45 amino acids amino acid <Unknown>linear protein 9 Glu Glu Ala Arg Phe Leu Thr Tyr Ser Met Trp Pro Leu SerPhe Le 1 5 10 15 Ser Pro Ala Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile GlyPro Gl 20 25 30 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu Ser 3540 45 45 amino acids amino acid <Unknown> linear protein 10 Glu Ala AsnArg Leu Val Thr Phe Lys Asp Trp Pro Asn Pro Asn Il 1 5 10 15 Thr Pro GlnAla Leu Ala Lys Ala Gly Phe Tyr Tyr Leu Asn Arg Le 20 25 30 Asp His ValLys Cys Val Trp Cys Asn Gly Val Ile Ala 35 40 45 45 amino acids aminoacid <Unknown> linear protein 11 Glu Glu Val Arg Leu Asn Thr Phe Glu LysTrp Pro Val Ser Phe Le 1 5 10 15 Ser Pro Glu Thr Met Ala Lys Asn Gly PheTyr Tyr Leu Gly Arg Se 20 25 30 Asp Glu Val Arg Cys Ala Phe Cys Lys ValGlu Ile Met 35 40 45 45 amino acids amino acid <Unknown> linear protein12 Lys Ala Ala Arg Leu Gly Thr Tyr Thr Asn Trp Pro Val Gln Phe Le 1 5 1015 Glu Pro Ser Arg Met Ala Ala Ser Gly Phe Tyr Tyr Leu Gly Arg Gl 20 2530 Asp Glu Val Arg Cys Ala Phe Cys Lys Val Glu Ile Thr 35 40 45 47 aminoacids amino acid <Unknown> linear protein 13 Glu Glu Ala Arg Leu Ala SerPhe Arg Asn Trp Pro Phe Tyr Val Gl 1 5 10 15 Gly Ile Ser Pro Cys Val LeuSer Glu Ala Gly Phe Val Phe Thr Gl 20 25 30 Lys Gln Asp Thr Val Gln CysPhe Ser Cys Gly Gly Cys Leu Gly 35 40 45 45 amino acids amino acid<Unknown> linear protein 14 Glu Ala Asn Arg Leu Val Thr Phe Lys Asp TrpPro Asn Pro Asn Il 1 5 10 15 Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe TyrTyr Leu Asn Arg Le 20 25 30 Asp His Val Lys Cys Val Trp Cys Asn Gly ValIle Ala 35 40 45 46 amino acids amino acid <Unknown> linear protein 15Glu Glu Ala Arg Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr Ala Hi 1 5 10 15Leu Thr Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Il 20 25 30Gly Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys 35 40 45 46amino acids amino acid <Unknown> linear protein 16 Glu Glu Ala Arg LeuLys Ser Phe Gln Asn Trp Pro Asp Tyr Ala Hi 1 5 10 15 Leu Thr Pro Arg GluLeu Ala Ser Ala Gly Leu Tyr Tyr Thr Gly Al 20 25 30 Asp Asp Gln Val GlnCys Phe Cys Cys Gly Gly Lys Leu Glu 35 40 45 45 amino acids amino acid<Unknown> linear protein 17 Glu Asn Ala Arg Leu Leu Thr Phe Gln Thr TrpPro Leu Thr Phe Le 1 5 10 15 Ser Pro Thr Asp Leu Ala Arg Ala Gly Phe TyrTyr Thr Gly Pro Gl 20 25 30 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly LysLeu Ser 35 40 45 45 amino acids amino acid <Unknown> linear protein 18Glu Glu Ala Arg Phe Leu Thr Tyr His Met Trp Pro Leu Thr Phe Le 1 5 10 15Ser Pro Ser Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gl 20 25 30Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu Ser 35 40 45 46 aminoacids amino acid <Unknown> linear protein 19 Glu Glu Ala Arg Leu Lys SerPhe Gln Asn Trp Pro Asp Tyr Ala Hi 1 5 10 15 Leu Thr Pro Arg Glu Leu AlaSer Ala Gly Leu Tyr Tyr Thr Gly Il 20 25 30 Gly Asp Gln Val Gln Cys PheCys Cys Gly Gly Lys Leu Lys 35 40 45 45 amino acids amino acid <Unknown>linear protein 20 Glu Ala Asn Arg Leu Val Thr Phe Lys Asp Trp Pro AsnPro Asn Il 1 5 10 15 Thr Pro Gln Ala Leu Ala Lys Ala Gly Phe Tyr Tyr LeuAsn Arg Le 20 25 30 Asp His Val Lys Cys Val Trp Cys Asn Gly Val Ile Ala35 40 45 50 amino acids amino acid <Unknown> linear protein 21 Tyr ValGly Ile Gly Asp Lys Val Lys Cys Phe His Cys Asp Gly Gl 1 5 10 15 Leu ArgAsp Trp Glu Pro Gly Asp Asp Pro Trp Glu Glu His Ala Ly 20 25 30 Trp PhePro Arg Cys Glu Phe Leu Leu Leu Ala Lys Gly Gln Glu Ty 35 40 45 Val Ser50 50 amino acids amino acid <Unknown> linear protein 22 Tyr Val Asp ArgAsn Asp Asp Val Lys Cys Phe Cys Cys Asp Gly Gl 1 5 10 15 Leu Arg Cys TrpGlu Pro Gly Asp Asp Pro Trp Ile Glu His Ala Ly 20 25 30 Trp Phe Pro ArgCys Glu Phe Leu Ile Arg Met Lys Gly Gln Glu Ph 35 40 45 Val Asp 50 50amino acids amino acid <Unknown> linear protein 23 Tyr Gln Lys Ile GlyAsp Gln Val Arg Cys Phe His Cys Asn Ile Gl 1 5 10 15 Leu Arg Ser Trp GlnLys Glu Asp Glu Pro Trp Phe Glu His Ala Ly 20 25 30 Trp Ser Pro Lys CysGln Phe Val Leu Leu Ala Lys Gly Pro Ala Ty 35 40 45 Val Ser 50 49 aminoacids amino acid <Unknown> linear protein 24 Tyr Thr Gly Tyr Gly Asp AsnThr Lys Cys Phe Tyr Cys Asp Gly Gl 1 5 10 15 Leu Lys Asp Trp Glu Pro GluAsp Val Pro Trp Glu Gln His Val Ar 20 25 30 Trp Phe Asp Arg Cys Ala TyrVal Gln Leu Val Lys Gly Arg Asp Ty 35 40 45 Val 49 amino acids aminoacid <Unknown> linear protein 25 Tyr Thr Gly Gln Gly Asp Lys Thr Arg CysPhe Cys Cys Asp Gly Gl 1 5 10 15 Leu Lys Asp Trp Glu Pro Asp Asp Ala ProTrp Gln Gln His Ala Ar 20 25 30 Trp Tyr Asp Arg Cys Glu Tyr Val Leu LeuVal Lys Gly Arg Asp Ph 35 40 45 Val 50 amino acids amino acid <Unknown>linear protein 26 Tyr Thr Gly Ile Lys Asp Ile Val Gln Cys Phe Ser CysGly Gly Cy 1 5 10 15 Leu Glu Lys Trp Gln Glu Gly Asp Asp Pro Leu Asp AspHis Thr Ar 20 25 30 Cys Phe Pro Asn Cys Pro Phe Leu Gln Asn Met Lys SerSer Ala Gl 35 40 45 Val Thr 50 50 amino acids amino acid <Unknown>linear protein 27 Tyr Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His CysAsn Ile Gl 1 5 10 15 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe GluHis Ala Ly 20 25 30 Trp Ser Pro Lys Cys Gln Phe Val Leu Leu Ala Lys GlyPro Ser Ty 35 40 45 Val Ser 50 50 amino acids amino acid <Unknown>linear protein 28 Ala Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His CysGly Gly Gl 1 5 10 15 Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu GlnHis Ala Ly 20 25 30 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu Gln Lys GlyGln Glu Ty 35 40 45 Ile Asn 50 50 amino acids amino acid <Unknown>linear protein 29 Ala Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His CysGly Gly Gl 1 5 10 15 Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu GlnHis Ala Ly 20 25 30 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Asp Glu Lys GlyGln Glu Ty 35 40 45 Ile Asn 50 50 amino acids amino acid <Unknown>linear protein 30 Tyr Val Gly Asn Ser Asp Asp Val Lys Cys Phe Cys CysAsp Gly Gl 1 5 10 15 Leu Arg Cys Trp Glu Ser Gly Asp Asp Pro Trp Val GlnHis Ala Ly 20 25 30 Trp Phe Pro Arg Cys Glu Tyr Leu Ile Arg Ile Lys GlyGln Glu Ph 35 40 45 Ile Arg 50 50 amino acids amino acid <Unknown>linear protein 31 Tyr Val Gly Arg Asn Asp Asp Val Lys Cys Phe Gly CysAsp Gly Gl 1 5 10 15 Leu Arg Cys Trp Glu Ser Gly Asp Asp Pro Trp Val GluHis Ala Ly 20 25 30 Trp Phe Pro Arg Cys Glu Phe Leu Ile Arg Met Lys GlyGln Glu Ph 35 40 45 Val Asp 50 50 amino acids amino acid <Unknown>linear protein 32 Ala Leu Gly Glu Gly Asp Lys Val Lys Cys Phe His CysGly Gly Gl 1 5 10 15 Leu Thr Asp Trp Lys Pro Ser Glu Asp Pro Trp Glu GlnHis Ala Ly 20 25 30 Trp Tyr Pro Gly Cys Lys Tyr Leu Leu Glu Gln Lys GlyGln Glu Ty 35 40 45 Ile Asn 50 50 amino acids amino acid <Unknown>linear protein 33 Tyr Gln Lys Ile Gly Asp Gln Val Arg Cys Phe His CysAsn Ile Gl 1 5 10 15 Leu Arg Ser Trp Gln Lys Glu Asp Glu Pro Trp Phe GluHis Ala Ly 20 25 30 Trp Ser Pro Lys Cys Gln Phe Val Leu Leu Ala Lys GlyPro Ala Ty 35 40 45 Val Ser 50 142 amino acids amino acid <Unknown>linear protein 34 Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro PheLeu Lys As 1 5 10 15 His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu GluGly Cys Al 20 25 30 Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile HisCys Pro Th 35 40 45 Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys PheLys Glu Le 50 55 60 Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu HisLys Lys Hi 65 70 75 80 Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys GlnPhe Glu Glu Le 85 90 95 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu ArgAla Lys Asn Ly 100 105 110 Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys GluPhe Glu Glu Thr Al 115 120 125 Lys Lys Val Arg Arg Ala Ile Glu Gln LeuAla Ala Met Asp 130 135 140 14796 base pairs nucleic acid single linearDNA (genomic) 35 TCTAGACATG CGGATATATT CAAGCTGGGC ACAGCACAGC AGCCCCACCCCAGGCAGCTT 60 GAAATCAGAG CTGGGGTCCA AAGGGACCAC ACCCCGAGGG ACTGTGTGGGGGTCGGGGC 120 CACAGGCCAC TGCTTCCCCC CGTCTTTCTC AGCCATTCCT GAAGTCAGCCTCACTCTGC 180 TCTCAGGGAT TTCAAATGTG CAGAGACTCT GGCACTTTTG TAGAAGCCCCTTCTGGTCC 240 AACTTACACC TGGATGCTGT GGGGCTGCAG CTGCTGCTCG GGCTCGGGAGGATGCTGGG 300 GCCCGGTGCC CATGAGCTTT TGAAGCTCCT GGAACTCGGT TTTGAGGGTGTTCAGGTCC 360 GGTGGACACC TGGGCTGTCC TTGTCCATGC ATTTGATGAC ATTGTGTGCAGAAGTGAAA 420 GGAGTTAGGC CGGGCATGCT GGCTTATGCC TGTAATCCCA GCACTTTGGGAGGCTGAGG 480 GGGTGGATCA CGAGGTCAGG AGTTCAATAC CAGCCTGGCC AAGATGGTGAAACCCCGTC 540 CTACTAAAAA TACAAAAAAA TTAGCCGGGC ATGGTGGCGG GCGCATGTAATCCCAGCTA 600 TGGGGGGGCT GAGGCAGAGA ATTGCTGGAA CCCAGGAGAT GGAGGTTGCAGTGAGCCAA 660 ATTGTGCCAC TGCACTGCAC TCCAGCCTGG CGACAGAGCA AGACTCTGTCTCAAAAAAA 720 AAAAAAAAAG TGAAAAGGAG TTGTTCCTTT CCTCCCTCCT GAGGGCAGGCAACTGCTGC 780 GTTGCCAGTG GAGGTGGTGC GTCCTTGGTC TGTGCCTGGG GGCCACCCCAGCAGAGGCC 840 TGGTGGTGCC AGGGCCCGGT TAGCGAGCCA ATCAGCAGGA CCCAGGGGCGACCTGCCAA 900 GTCAACTGGA TTTGATAACT GCAGCGAAGT TAAGTTTCCT GATTTTGATGATTGTGTTG 960 GGTTGTGTAA GAGAATGAAG TATTTCGGGG TAGTATGGTA ATGCCTTCAACTTACAAA 1020 GTTCAGGTAA ACCACCCATA TACATACATA TACATGCATG TGATATATACACATACAG 1080 ATGTGTGTGT GTTCACATAT ATGAGGGGAG AGAGACTAGG GGAGAGAAAGTAGGTTGG 1140 AGAGGGAGAG AGAAAGGAAA ACAGGAGACA GAGAGAGAGC GGGGAGTAGAGAGAGGGA 1200 GGGTAAGAGA GGGAGAGGAG GAGAGAAAGG GAGGAAGAAG CAGAGAGTGAATGTTAAA 1260 AAACAGGCAA AACATAAACA GAAAATCTGG GTGAAGGGTA TATGAGTATTCTTTGTAC 1320 TTCTTGCAAT TATCTTTTAT TTAAATTGAC ATCGGGCCGG GCGCAGTGGCTCACATCT 1380 AATCCCAGCA CTTTGGGAGG CCGAGGCAGG CAGATCACTT GAGGTCAGGAGTTTGAGA 1440 AGCCTGGCAA ACATGGTGAA ACCCCATCTC TACTAAAAAT ACAAAAATTAGCCTGGTG 1500 GTGGTGCATG CCTTTAATCT CAGCTACTCG GGAGGCTGAG GCAGGAGAATCGCTTGAA 1560 CGTGGCGGGG AGGAGGTTGC AGTGAGCTGA GATCATGCCA CTGCACTCCAGCCTGGGC 1620 TAGAGCGAGA CTCAGTTTCA AATAAATAAA TAAACATCAA AATAAAAAGTTACTGTAT 1680 AAGAATGGGG GCGGGGTGGG AGGGGTGGGG AGAGGTTGCA AAAATAAATAAATAAATA 1740 TAAACCCCAA AATGAAAAAG ACAGTGGAGG CACCAGGCCT GCGTGGGGCTGGAGGGCT 1800 TAAGGCCAGG CCTCTTATCT CTGGCCATAG AACCAGAGAA GTGAGTGGATGTGATGCC 1860 GCTCCAGAAG TGACTCCAGA ACACCCTGTT CCAAAGCAGA GGACACACTGATTTTTTT 1920 TAATAGGCTG CAGGACTTAC TGTTGGTGGG ACGCCCTGCT TTGCGAAGGGAAAGGAGG 1980 TTTGCCCTGA GCACAGGCCC CCACCCTCCA CTGGGCTTTC CCCAGCTCCCTTGTCTTC 2040 ATCACGGTAG TGGCCCAGTC CCTGGCCCCT GACTCCAGAA GGTGGCCCTCCTGGAAAC 2100 AGGTCGTGCA GTCAACGATG TACTCGCCGG GACAGCGATG TCTGCTGCACTCCATCCC 2160 CCCTGTTCAT TTGTCCTTCA TGCCCGTCTG GAGTAGATGC TTTTTGCAGAGGTGGCAC 2220 TGTAAAGCTC TCCTGTCTGA CTTTTTTTTT TTTTTTAGAC TGAGTTTTGCTCTTGTTG 2280 TAGGCTGGAG TGCAATGGCA CAATCTCAGC TCACTGCACC CTCTGCCTCCCGGGTTCA 2340 CGATTCTCCT GCCTCAGCCT CCCGAGTAGT TGGGATTACA GGCATGCACCACCACGCC 2400 GCTAATTTTT GTATTTTTAG TAGAGACAAG GTTTCACCGT GATGGCCAGGCTGGTCTT 2460 ACTCCAGGAC TCAAGTGATG CTCCTGCCTA GGCCTCTCAA AGTGTTGGGATTACAGGC 2520 GAGCCACTGC ACCCGGCCTG CACGCGTTCT TTGAAAGCAG TCGAGGGGGCGCTAGGTG 2580 GGCAGGGACG AGCTGGCGCG GCGTCGCTGG GTGCACCGCG ACCACGGGCAGAGCCACG 2640 GCGGGAGGAC TACAACTCCC GGCACACCCC GCGCCGCCCC GCCTCTACTCCCAGAAGG 2700 GCGGGGGGTG GACCGCCTAA GAGGGCGTGC GCTCCCGACA TGCCCCGCGGCGCGCCAT 2760 ACCGCCAGAT TTGAATCGCG GGACCCGTTG GCAGAGGTGG CGGCGGCGGCATGGGTGC 2820 CGACGTTGCC CCCTGCCTGG CAGCCCTTTC TCAAGGACCA CCGCATCTCTACATTCAA 2880 ACTGGCCCTT CTTGGAGGGC TGCGCCTGCA CCCCGGAGCG GGTGAGACTGCCCGGCCT 2940 TGGGGTCCCC CACGCCCGCC TTGCCCTGTC CCTAGCGAGG CCACTGTGACTGGGCCTC 3000 GGGTACAAGC CGCCCTCCCC TCCCCGTCCT GTCCCCAGCG AGGCCACTGTGGCTGGGC 3060 CTTGGGTCCA GGCCGGCCTC CCCTCCCTGC TTTGTCCCCA TCGAGGCCTTTGTGGCTG 3120 CCTCGGGGTT CCGGGCTGCC ACGTCCACTC ACGAGCTGTG CTGTCCCTTGCAGATGGC 3180 AGGCTGGCTT CATCCACTGC CCCACTGAGA ACGAGCCAGA CTTGGCCCAGTGTTTCTT 3240 GCTTCAAGGA GCTGGAAGGC TGGGAGCCAG ATGACGACCC CATGTAAGTCTTCTCTGG 3300 AGCCTCGATG GGCTTTGTTT TGAACTGAGT TGTCAAAAGA TTTGAGTTGCAAAGACAC 3360 AGTATGGGAG GGTTGCTTTC CACCCTCATT GCTTCTTAAA CAGCTGTTGTGAACGGAT 3420 CTCTCTATAT GCTGGTGCCT TGGTGATGCT TACAACCTAA TTAAATCTCATTTGACCA 3480 ATGCCTTGGG GTGGACGTAA GATGCCTGAT GCCTTTCATG TTCAACAGAATACATCAG 3540 GACCCTGTTG TTGTGAACTC CCAGGAATGT CCAAGTGCTT TTTTTGAGATTTTTTAAA 3600 ACAGTTTAAT TGAAATATAA CCTACACAGC ACAAAAATTA CCCTTTGAAAGTGTGCAC 3660 CACACTTTCG GAGGCTGAGG CGGGCGGATC ACCTGAGGTC AGGAGTTCAAGACCTGCC 3720 GCCAACTTGG CGAAACCCCG TCTCTACTAA AAATACAAAA ATTAGCCGGGCATGGTAG 3780 CACGCCCGTA ATCCCAGCTA CTCGGGAGGC TAAGGCAGGA GAATCGCTTGAACCTGGG 3840 GCGGAGGTTG CAGTGAGCCG AGATTGTGCC AATGCACTCC AGCCTCGGCGACAGAGCG 3900 ACTCCGTCAT AAAAATAAAA AATTGAAAAA AAAAAAAGAA AGAAAGCATATACTTCAG 3960 TTGTTCTGGA TTTTTTTCTT CAAGATGCCT AGTTAATGAC AATGAAATTCTGTACTCG 4020 TGGTATCTGT CTTTCCACAC TGTAATGCCA TATTCTTTTC TCACCTTTTTTTCTGTCG 4080 TTCAGTTGCT TCCACAGCTT TAATTTTTTT CCCCTGGAGA ATCACCCCAGTTGTTTTT 4140 TTTTGGCCAG AAGAGAGTAG CTGTTTTTTT TCTTAGTATG TTTGCTATGGTGGTTATA 4200 GCATCCCCGT AATCACTGGG AAAAGATCAG TGGTATTCTT CTTGAAAATGAATAAGTG 4260 ATGATATTTT CAGATTAGAG TTACAACTGG CTGTCTTTTT GGACTTTGTGTGGCCATG 4320 TTCATTGTAA TGCAGTTCTG GTAACGGTGA TAGTCAGTTA TACAGGGAGACTCCCCTA 4380 AGAAAATGAG AGTGTGAGCT AGGGGGTCCC TTGGGGAACC CGGGGCAATAATGCCCTT 4440 CTGCCCTTAA TCCTTACAGT GGGCCGGGCA CGGTGGCTTA CGCCTGTAATACCAGCAC 4500 TGGGAGGCCG AGGCGGGCGG ATCACGAGGT CAGGAGATCG AGACCATCTTGGCTAATA 4560 GTGAAACCCC GTCTCCACTA AAAATACAAA AAATTAGCCG GGCGTGGTGGTGGGCGCC 4620 TAGTCCCAGC TACTCGGGAG GCTGAGGCAG GAGAATGGCG TGAACCCAGGAGGCGGAG 4680 TGCAGTGAGC CGAGATTGCA CCACTGCACT CCAGCCTGGG CGACAGAATGAGACTCCG 4740 TCAAAAAAAA AAAAAAAAGA AAAAAATCTT TACAGTGGAT TACATAACAATTCCAGTG 4800 ATGAAATTAC TTCAAACAGT TCCTTGAGAA TGTTGGAGGG ATTTGACATGTAATTCCT 4860 GGACATATAC CATGTAACAC TTTTCCAACT AATTGCTAAG GAAGTCCAGATAAAATAG 4920 ACATTAGCCA CACAGATGTG GGGGGAGATG TCCACAGGGA GAGAGAAGGTGCTAAGAG 4980 GCCATATGGG AATGTGGCTT GGGCAAAGCA CTGATGCCAT CAACTTCAGACTTGACGT 5040 TACTCCTGAG GCAGAGCAGG GTGTGCCTGT GGAGGGCGTG GGGAGGTGGCCCGTGGGG 5100 TGGACTGCCG CTTTAATCCC TTCAGCTGCC TTTCCGCTGT TGTTTTGATTTTTCTAGA 5160 GGAACATAAA AAGCATTCGT CCGGTTGCGC TTTCCTTTCT GTCAAGAAGCAGTTTGAA 5220 ATTAACCCTT GGTGAATTTT TGAAACTGGA CAGAGAAAGA GCCAAGAACAAAATTGTA 5280 TATTGGGAAT AAGAACTGCT CAAACCCTGT TCAATGTCTT TAGCACTAAACTACCTAG 5340 CCTCAAAGGG ACTCTGTGTT TTCCTCAGGA AGCATTTTTT TTTTTTTTCTGAGATAGA 5400 TTCACTCTTG TTGCCCAGGC TGGAGTGCAA TGGTGCAATC TTGGCTCACTGCAACCTC 5460 CCTCTCGGGT TCAAGTGATT CTCCTGCCTC AGCCTCCCAA GTAACTGGGATTACAGGG 5520 GTGCCACCAC ACCCAGCTAA TTTTTGTATT TTTAGTAGAG ATGGGGTTTCACCACATT 5580 CCAGGCTGGT CTTGAACTCC TGACCTCGTG ATTCGCCCAC CTTGGCCTCCCAAAGTGC 5640 GGATTACAGG CGTGAACCAC CACGCCTGGC TTTTTTTTTT TTGTTCTGAGACACAGTT 5700 ACTCTGTTAC CCAGGCTGGA GTAGGGTGGC CTGATCTCGG ATCACTGCAACCTCCGCC 5760 CTGGGCTCAA GTGATTTGCC TGCTTCAGCC TCCCAAGTAG CCGAGATTACAGGCATGT 5820 CACCACACCC AGGTAATTTT TGTATTTTTG GTAGAGACGA GGTTTCACCATGTTGGCC 5880 GCTGGTTTTG AACTCCTGAC CTCAGGTGAT CCACCCGCCT CAGCCTCCCAAAGTGCTG 5940 ATTATAGGTG TGAGCCACCA CACCTGGCCT CAGGAAGTAT TTTTATTTTTAAATTTAT 6000 ATTTATTTGA GATGGAGTCT TGCTCTGTCG CCCAGGCTAG AGTGCAGCGACGGGATCT 6060 GCTCACTGCA AGCTCCGCCC CCCAGGTTCA AGCCATTCTC CTGCCTCAGCCTCCCGAG 6120 GCTGGGACTA CAGGCGCCCG CCACCACACC CGGCTAATTT TTTTGTATTTTTAGTAGA 6180 CGGGTTTTCA CCGTGTTAGC CAGGAGGGTC TTGATCTCCT GACCTCGTGATCTGCCTG 6240 TCGGCCTCCC AAAGTGCTGG GATTACAGGT GTGAGCCACC ACACCCGGCTATTTTTAT 6300 TTTTGAGACA GGGACTCACT CTGTCACCTG GGCTGCAGTG CAGTGGTACACCATAGCT 6360 CTGCAGCCTC GAACTCCTGA GCTCAAGTGA TCCTCCCACC TCATCCTCACAAGTAATT 6420 GACTACAGGT GCACCCCACC ATGCCCACCT AATTTATTTA TTTATTTATTTATTTATT 6480 CATAGAGATG AGGGTTCCCT GTGTTGTCCA GGCTGGTCTT GAACTCCTGAGCTCACGG 6540 TCCTTTTGCC TGGGCCTCCC AAAGTGCTGA GATTACAGGC ATGAGCCACCGTGCCCAG 6600 AGGAATCATT TTTAAAGCCC CTAGGATGTC TGTGTGATTT TAAAGCTCCTGGAGTGTG 6660 CGGTATAAGT ATATACCGGT ATAAGTAAAT CCCACATTTT GTGTCAGTATTTACTAGA 6720 CTTAGTCATT TATCTGAAGT TGAAATGTAA CTGGGCTTTA TTTATTTATTTATTTATT 6780 TTTATTTTTA ATTTTTTTTT TTGAGACGAG TCTCACTTTG TCACCCAGGCTGGAGTGC 6840 TGGCACGATC TCGGCTCACT GCAACCTCTG CCTCCCGGGG TCAAGCGATTCTCCTGCC 6900 AGCCTCCCGA GTAGCTGGGA CTACAGGCAC GCACCACCAT GCCTGGCTAATTTTTGTA 6960 TTTAGTAGAC GGGGTTTCAC CATGCTGGCC AAGCTGGTCT CAAACTCCTGACCTTGTG 7020 CTGCCCGCTT TAGCCTCCCA GAGTGCTGGG ATTACAGGCA TGAGCCACCATGCGTGGT 7080 TTTTAAAATT TTTTGATTTT TTTTTTTTTT GAGACAGAGC CTTGCTCTGTCGCCCAGG 7140 GGAGTGCAGT GGCACGATCT CAGCTCACTA CAAGCTCCGC CTCCCGGGTTCACGCCAT 7200 TTCTGCCTCA GCCTCCTGAG TAGCTGGGAC TACAGGTGCC CACCACCACGCCTGGCTA 7260 TTTTTTTGGT ATTTTTATTA GAGACAAGGT TTCATCATGT TGGCCAGGCTGGTCTCAA 7320 TCCTGACCTC AAGTGATCTG CCTGCCTCGG CCTCCCAAAG CGCTGAGATTACAGGTGT 7380 TCTACTGCGC CAGGCCTGGG CGTCATATAT TCTTATTTGC TAAGTCTGGCAGCCCCAC 7440 AGAATAAGTA CTGGGGGATT CCATATCCTT GTAGCAAAGC CCTGGGTGGAGAGTCAGG 7500 ATGTTGTAGT TCTGTCTCTG CCACTTGCAG ACTTTGAGTT TAAGCCAGTCGTGCTCAT 7560 TTTCCTTGCT AAATAGAGGT TAGACCCCCT ATCCCATGGT TTCTCAGGTTGCTTTTCA 7620 TTGAAAATTG TATTCCTTTG TAGAGATCAG CGTAAAATAA TTCTGTCCTTATATGTGG 7680 TTATTTTAAT TTGAGACAGA GTGTCACTCA GTCGCCCAGG CTGGAGTGTGGTGGTGCG 7740 CTTGGCTCAC TGCGACCTCC ACCTCCCAGG TTCAAGCGAT TCTCGTGCCTCAGGCTCC 7800 AGTAGCTGAG ATTATAGGTG TGTGCCACCA GGCCCAGCTA ACTTTTGTATTTTTAGTA 7860 GACAGGGTTT TGCCATGTTG GCTAAGCTGG TCTCGAACTC CTGGCCTCAAGTGATCTG 7920 CGCCTTGGCA TCCCAAAGTG CTGGGATTAC AGGTGTGAAC CACCACACCTGGCCTCAA 7980 TAGTGGCTTT TAAGTGCTAA GGACTGAGAT TGTGTTTTGT CAGGAAGAGGCCAGTTGT 8040 GTGAAGCATG CTGTGAGAGA GCTTGTCACC TGGTTGAGGT TGTGGGAGCTGCAGCGTG 8100 AACTGGAAAG TGGGCTGGGG ATCATCTTTT TCCAGGTCAG GGGTCAGCCAGCTTTTCT 8160 AGCGTGCCAT AGACCATCTC TTAGCCCTCG TGGGTCAGAG TCTCTGTTGCATATTGTC 8220 TTGTTGTTTT TCACAACCTT TTAGAAACAT AAAAAGCATT CTTAGCCCGTGGGCTGGA 8280 AAAAAAGGCC ATGACGGGCT GTATGGATTT GGCCCAGCAG GCCCTTGCTTGCCAAGCC 8340 GTTTTAGACA AGGAGCAGCT TGTGTGCCTG GAACCATCAT GGGCACAGGGGAGGAGCA 8400 GTGGATGTGG AGGTGTGAGC TGGAAACCAG GTCCCAGAGC GCTGAGAAAGACAGAGGG 8460 TTTGCCCTTG CAAGTAGAGC AACTGAAATC TGACACCATC CAGTTCCAGAAAGCCCTG 8520 GTGCTGGTGG ACGCTGCGGG GTGCTCCGCT CTAGGGTTAC AGGGATGAAGATGCAGTC 8580 GTAGGGGGAG TCCACTCACC TGTTGGAAGA TGTGATTAAG AAAAGTAGACTTTCAGGG 8640 GGGCATGGTG GCTCACGCCT GTAATCCCAG CACTTTGGGA GGCCGAGGCGGGTGGATC 8700 GAGGTCAGGA GATCGAGACC ATCCTGGCTA ACATGGTGAA ACCCCGTCTTTACTAAAA 8760 ACAAAAAATT AGCTGGGCGT GGTGGCGGGC GCCTGTAGTC CCAGCTACTCGGGAGGCT 8820 GGCAGGAGAA TGGCGTGAAC CTGGGAGGTG GAGCTTGCTG TGAGCCGAGATCGCGCCA 8880 GCACTCCAGC CTGGGCGACA GAGCGAGACT CCGTCTCAAA AAAAAAAAAAAAAGTAGG 8940 TTCATGATGT GTGAGCTGAA GGCGCAGTAG GCAGAAGTAG AGGCCTCAGTCCCTGCAG 9000 GACCCCTCGG TCTCTATCTC CTGATAGTCA GACCCAGCCA CACTGGAAAGAGGGGAGA 9060 TTACAGCCTG CGAGAAAAGT AGGGAGATTT AAAAACTGCT TGGCTTTTATTTTGAACT 9120 TTTTTTTGTT TGTTTGTTTT CCCCAATTCA GAATACAGAA TACTTTTATGGATTTGTT 9180 TATTACTTTA ATTTTGAAAC AATATAATCT TTTTTTTGTT GTTTTTTTGAGACAGGGT 9240 TACTCTGTCA CCCAGGCTGA GTGCAGTGGT GTGATCTTGG CTCACCTCAGCCTCGACC 9300 CTGGGCTCAA ATGATTCTCC CACCTCAGCT TCCCAAGTAG CTGGGACCACAGGTGCGT 9360 GTTGCGCTAT ACAAATCCTG AAGACAAGGA TGCTGTTGCT GGTGATGCTGGGGATTCC 9420 AGATCCCAGA TTTGATGGCA GGATGCCCCT GTCTGCTGCC TTGCCAGGGTGCCAGGAG 9480 CGCTGCTGTG GAAGCTGAGG CCCGGCCATC CAGGGCGATG CATTGGGCGCTGATTCTT 9540 TCCTGCTGCT GCCTCGGTGC TTAGCTTTTG AAACAATGAA ATAAATTAGAACCAGTGT 9600 AAATCGATCA GGGAATAAAT TTAATGTGGA AATAAACTGA ACAACTTAGTTCTTCATA 9660 AGTTTACTTG GTAAATACTT GTGATGAGGA CAAAACGAAG CACTAGAAGGAGAGGCGA 9720 TGTAGACCTG GGTGGCAGGA GTGTTTTGTT TGTTTTCTTT GGCAGGGTCTTGCTCTGT 9780 CTCAGGCTGG AGTACAGTGG CACAATCACA GCTCACTATA GCCTCGACCTCCTGGACT 9840 AGCAATCCTC CTGCCTCAGC CTCCCAGTAG CTGGGACTAC AGGCGCATGCCACCATGC 9900 GGCTAATTTT AAATTTTTTT TTTTCTCTTT TTTGAGATGG AATCTCACTCTGTCGCCC 9960 GCTGGAGTGC AGTGGCGTGA TCTCGGCTGA CGGCAAGCTC CGCCTCCCAGGTTCACT 10020 TTCGCCTGCC TCAGCCTCCC AAGTAGCTGG GACTACAGGC GCTGGGATTACAAACCC 10080 CCCAAAGTGC TGGGATTACA GGCGTGAGCC ACTGCACCCG GCCTGTTTTGTCTTTCA 10140 GCAAGAGTTG TGTTTGCTTC GCCCCTACCT TTAGTGGAAA AATGTATAAAATGGAGA 10200 TGACCTCCAC ATTGGGGTGG TTAAATTATA GCATGTATGC AAAGGAGCTTCGCTAAT 10260 AGGCTTTTTT GAAAGAGAAG AAACTGAATA ATCCATGTGT GTATATATATTTTAAAA 10320 ATGGTCATCT TTCCATATCA GTAAAGCTGA GGCTCCCTGG GACTGCAGAGTTGTCCA 10380 CAGTCCATTA TAAGTGCGCT GCTGGGCCAG GTGCAGTGGC TTGTGCCTGAATCCCAG 10440 TTTGGGAGGC CAAGGCAGGA GGATTCATTG AGCCCAGGAG TTTTGAGGCGAGCCTGG 10500 ATGTGGCCAG ACCTCATCTC TTCAAAAAAT ACACAAAAAA TTAGCCAGGCATGGTGG 10560 GTGCCTGTAG TCTCAGCTAC TCAGGAGGCT GAGGTGGGAG GATCACTTTGAGCCTTG 10620 GTCAAAGCTG CAGTAAGCCA TGATCTTGCC ACTGCATTCC AGCCTGGATGACAGAGC 10680 ACCCTGTCTC TAAAAAAAAA AAAAACCAAA CGGTGCACTG TTTTCTTTTTTCTTATC 10740 TTATTATTTT TAAATTAAAT TTTCTTTTAA TAATTTATAA ATTATAAATTTATATTA 10800 AATGACAAAT TTTTATTACT TATACATGAG GTAAAACTTA GGATATATAAAGTACAT 10860 GAAAAGTAAT TTTTTGGCTG GCACAGTGGC TCACACCTGT AATCCCAGCACTTTGGG 10920 CCGTGGCGGG CAGATCACAT GAGATCATGA GTTCGAGACC AACCTGACCAACATGGA 10980 ACCCCATCTC TACTAAAAAT ACAAAATTAG CCGGGGTGGT GGCGCATGCCTGTAATC 11040 GCTACTCGGG AGGCTGAGGC AGGAGAATCT CTTGAACCCG GGAGGCAGAGGTTGCGG 11100 GCCAAGATCG TGCCTTTGCA CACCAGCCTA GGCAACAAGA GCGAAAGTCCGTCTCAA 11160 AAAAGTAATT TTTTTTAAGT TAACCTCTGT CAGCAAACAA ATTTAACCCAATAAAGG 11220 TTGTTTTTTA ATGTAGTAGA GGAGTTAGGG TTTATAAAAA ATATGGTAGGGAAGGGG 11280 CCTGGATTTG CTAATGTGAT TGTCATTTGC CCCTTAGGAG AGAGCTCTGTTAGCAGA 11340 AAAAAATTGG AAGCCAGATT CAGGGAGGGA CTGGAAGCAA AAGAATTTCTGTTCGAG 11400 GAGCCTGATG TTTGCCAGGG TCTGTTTAAC TGGACATGAA GAGGAAGGCTCTGGACT 11460 CTCCAGGAGT TTCAGGAGAA AGGTAGGGCA GTGGTTAAGA GCAGAGCTCTGCCTAGA 11520 GCTGGGGTGC CTAGACTAGC TGGGGTGCCC AGACTAGCTG GGGTGCCTAGACTAGCT 11580 TACTTTGAGT GGCTCCTTCA GCCTGGACCT CGGTTTCCTC ACCTGTATAGTAGAGAT 11640 GGAGCACCCA GCGCAGGATC ACTGTGAACA TAAATCAGTT AATGGAGGAAGCAGGTA 11700 TGGTGCTGGG TGCATACCAA GCACTCCGTC AGTGTTTCCT GTTATTCGATGATTAGG 11760 CAGCTTAAAC TAGAGGGAGT TGAGCTGAAT CAGGATGTTT GTCCCAGGTAGCTGGGA 11820 TGCCTAGCCC AGTGCCCAGT TTATTTAGGT GCTCTCTCAG TGTTCCCTGATTGTTTT 11880 CTTTGTCATC TTATCTACAG GATGTGACTG GGAAGCTCTG GTTTCAGTGTCATGTGT 11940 TTCTTTATTT CCAGGCAAAG GAAACCAACA ATAAGAAGAA AGAATTTGAGGAAACTG 12000 AGAAAGTGCG CCGTGCCATC GAGCAGCTGG CTGCCATGGA TTGAGGCCTCTGGCCGG 12060 TGCCTGGTCC CAGAGTGGCT GCACCACTTC CAGGGTTTAT TCCCTGGTGCCACCAGC 12120 CCTGTGGGCC CCTTAGCAAT GTCTTAGGAA AGGAGATCAA CATTTTCAAATTAGATG 12180 CAACTGTGCT CCTGTTTTGT CTTGAAAGTG GCACCAGAGG TGCTTCTGCCTGTGCAG 12240 GTGCTGCTGG TAACAGTGGC TGCTTCTCTC TCTCTCTCTC TTTTTTGGGGGCTCATT 12300 GCTGTTTTGA TTCCCGGGCT TACCAGGTGA GAAGTGAGGG AGGAAGAAGGCAGTGTC 12360 TTTGCTAGAG CTGACAGCTT TGTTCGCGTG GGCAGAGCCT TCCACAGTGAATGTGTC 12420 ACCTCATGTT GTTGAGGCTG TCACAGTCCT GAGTGTGGAC TTGGCAGGTGCCTGTTG 12480 CTGAGCTGCA GGTTCCTTAT CTGTCACACC TGTGCCTCCT CAGAGGACAGTTTTTTT 12540 GTTGTGTTTT TTTGTTTTTT TTTTTTGGTA GATGCATGAC TTGTGTGTGATGAGAGA 12600 GAGACAGAGT CCCTGGCTCC TCTACTGTTT AACAACATGG CTTTCTTATTTTGTTTG 12660 TGTTAATTCA CAGAATAGCA CAAACTACAA TTAAAACTAA GCACAAAGCCATTCTAA 12720 ATTGGGGAAA CGGGGTGAAC TTCAGGTGGA TGAGGAGACA GAATAGAGTGATAGGAA 12780 TCTGGCAGAT ACTCCTTTTG CCACTGCTGT GTGATTAGAC AGGCCCAGTGAGCCGCG 12840 CACATGCTGG CCGCTCCTCC CTCAGAAAAA GGCAGTGGCC TAAATCCTTTTTAAATG 12900 TGGCTCGATG CTGTGGGGGA CTGGCTGGGC TGCTGCAGGC CGTGTGTCTGTCAGCCC 12960 CTTCACATCT GTCACGTTCT CCACACGGGG GAGAGACGCA GTCCGCCCAGGTCCCCG 13020 TCTTTGGAGG CAGCAGCTCC CGCAGGGCTG AAGTCTGGCG TAAGATGATGGATTTGA 13080 GCCCTCCTCC CTGTCATAGA GCTGCAGGGT GGATTGTTAC AGCTTCGCTGGAAACCT 13140 GAGGTCATCT CGGCTGTTCC TGAGAAATAA AAAGCCTGTC ATTTCAAACACTGCTGT 13200 CCCTACTGGG TTTTTAAAAT ATTGTCAGTT TTTCATCGTC GTCCCTAGCCTGCCAAC 13260 CATCTGCCCA GACAGCCGCA GTGAGGATGA GCGTCCTGGC AGAGACGCAGTTGTCTC 13320 GCGCTTGCCA GAGCCACGAA CCCCAGACCT GTTTGTATCA TCCGGGCTCCTTCCGGG 13380 AAACAACTGA AAATGCACTT CAGACCCACT TATTTATGCC ACATCTGAGTCGGCCTG 13440 TAGACTTTTC CCTCTAAACT GGGAGAATAT CACAGTGGTT TTTGTTAGCAGAAAATG 13500 TCCAGCCTCT GTACTCATCT AAGCTGCTTA TTTTTGATAT TTGTGTCAGTCTGTAAA 13560 ATACTTCACT TTAATAACTG TTGCTTAGTA ATTGGCTTTG TAGAGAAGCTGGAAAAA 13620 GGTTTTGTCT TCAACTCCTT TGCATGCCAG GCGGTGATGT GGATCTCGGCTTCTGTG 13680 CTGTGCTGTG GGCAGGGCTG AGCTGGAGCC GCCCCTCTCA GCCCGCCTGCCACGGCC 13740 CCTTAAAGGC CATCCTTAAA ACCAGACCCT CATGGCTGCC AGCACCTGAAAGCTTCC 13800 ACATCTGTTA ATAAAGCCGT AGGCCCTTGT CTAAGCGCAA CCGCCTAGACTTTCTTT 13860 ATACATGTCC ACATGTCCAT TTTTCAGGTT CTCTAAGTTG GAGTGGAGTCTGGGAAG 13920 TGTGAATGAG GCTTCTGGGC TATGGGTGAG GTTCCAATGG CAGGTTAGAGCCCCTCG 13980 CAACTGCCAT CCTGGAAAGT AGAGACAGCA GTGCCCGCTG CCCAGAAGAGACCAGCA 14040 CAAACTGGAG CCCCCATTGC AGGCTGTCGC CATGTGGAAA GAGTAACTCACAATTGC 14100 TAAAGTCTCA TGTGGTTTTA TCTACTTTTT TTTTCTTTTT CTTTTTTTTTGAGACAA 14160 CTTGCCCTCC CAGGCTGGAG TGCAGTGGAA TGACCACAGC TCACCGCAACCTCAAAT 14220 TGCGTTCAAG TGAACCTCCC ACTTTAGCCT CCCAAGTAGC TGGGACTACAGGCGCAC 14280 ATCACACCCG GCTAATTGAA AAATTTTTTT TTTTGTTTAG ATGGAATCTCACTTTGT 14340 CCAGGCTGGT CTCAAACTCC TGGGCTCAAG TGATCATCCT GCTTCAGCGTCCGACTT 14400 GGTATTATAG GCGTGAGCCA CTGGGCCTGA CCTAGCTACC ATTTTTTAATGCAGAAA 14460 AGACTTGTAG AAATGAAATA ACTTGTCCAG GATAGTCGAA TAAGTAACTTTTAGAGC 14520 GATTTGAACC CAGGCAATCT GGCTCCAGAG CTGGGCCCTC ACTGCTGAAGGACACTG 14580 GCTTGGGAGG GTGGCTATGG TCGGCTGTCT GATTCTAGGG AGTGAGGGCTGTCTTTA 14640 CACCCCATTC CATTTTCAGA CAGCTTTGTC AGAAAGGCTG TCATATGGAGCTGACAC 14700 CCTCCCCAAG GCTTCCATAG ATCCTCTCTG TACATTGTAA CCTTTTATTTTGAAATG 14760 ATTCACAGGA AGTTGTAAGG CTAGTACAGG GGATCC 14796

What is claimed:
 1. A method of modulating apoptosis in a cell,comprising the step of adminstering to the cell an agent that modulatesthe amount or activity of Survivin in the cell.
 2. The method of claim1, wherein the level of apoptosis is increased by decreasing the amountor activity of Survivin in the cell.
 3. The method of claim 1, whereinthe level of apoptosis is decreased by increasing the amount or activityof Survivin in the cell.
 4. A method of inhibiting apoptosis in a cell,comprising the step of administering to the cell a Survivin polypeptide,Survivin polypeptide fragment or an apoptosis-inhibiting peptidomimeticthereof in an amount effective to inhibit apoptosis in the cell.
 5. Amethod of inhibiting apoptosis in a cell, comprising the step ofadministering to the cell a transgene encoding a Survivin polypeptide orSurvivin polypeptide fragment thereof, the transgene being effective tocause expression of the Survivin polypeptide or fragment thereof in anamount effective to inhibit apoptosis in the cell.
 6. A method ofincreasing apoptosis in a cell, comprising the step of administering tothe cell an agent that decreases the apoptosis-inhibiting activity ofthe Survivin polypeptide present in the cell, in an amount that iseffective to increase the level of apoptosis in the cell.
 7. The methodof claim 6, wherein said agent is a polypeptide comprising the sequenceEGWEPDDDPIEEHKKHSSGC, its conservatively substituted homologs or smallmolecule peptidomimetics thereof.
 8. A method of increasing the level ofapoptosis in a cell, comprising the step of administering to the cell anagent that increases the transcription of the sense strand of EPR-1, inan amount effect to inhibit the translation of mRNA encoding a Survivinpolypeptide, thereby decreasing the transcription of the Survivinpolypeptide and increasing the level of apoptosis in the cell.
 9. Anisolated nucleic acid molecule that encodes the amino acid sequencedepicted in FIG. 10, allelic variants of the amino acid sequence of FIG.10, and fragments thereof that are effective to inhibit apoptosis. 10.The isolated nucleic acid molecule of claim 9, wherein said nucleic acidmolecule is operably linked to one or more expression control elements.11. The isolated nucleic acid molecule of claim 9, wherein said nucleicacid molecule is included in a vector.
 12. An isolated nucleic acidmolecule that encodes a member of the Survivin family of proteins,wherein said nucleic acid molecule hybridizes to a nucleic acid moleculeof claim 9 under conditions of sufficient stringency to produce a clearsignal.
 13. A host cell transformed to contain the nucleic acid moleculeof claim
 9. 14. The host cell of claim 13, wherein said host is selectedfrom the group consisting of prokaryotic hosts and eukaryotic hosts. 15.A method for producing a Survivin protein comprising the step ofculturing a host transformed with the nucleic acid molecule of claim 11under conditions in which the Survivin protein is expressed.
 16. Themethod of claim 15, wherein said host is selected from the groupconsisting of prokaryotic hosts and eukaryotic hosts.
 17. An isolatedpolypeptide comprising the amino acid sequence depicted in FIG. 10,allelic variants thereof and fragments thereof that retain the abilityto inhibit cellular apoptosis.
 18. A polypeptide comprising the sequenceEGWEPDDDPIEEHKKHSSGC, its conservatively substituted homologs and smallmolecule peptidomimetics thereof.
 19. A monoclonal antibody that bindsto the polypeptide, allelic variants thereof and fragments thereof thatretain the ability to inhibit cellular apoptosis of claim
 17. 20. Themonoclonal antibody of claim 19 which has been humanized.
 21. A methodfor reducing the severity of a pathological state mediated by Survivincomprising the step of reducing Survivin expression or activity.
 22. Themethod of claim 21 wherein said pathological state is caused by abnormalcell growth.
 23. The method of claim 21 wherein said Survivin expressionis reduced by contacting affected cells with an RNA molecule that iscomplementary to a Survivin encoding mRNA molecule.
 24. The method ofclaim 21, wherein said Survivin activity is reduced by blocking theintracellular interaction of Survivin with a Survivin binding partnercomprising the step of contacting said Survivin with an agent thatblocks the binding of Survivin to said binding partner.
 25. The methodof claim 24 wherein said agent blocks the binding of said Survivin tosaid binding partner by selectively binding to Survivin.
 26. The methodof claim 25 wherein said agent blocks the binding of said Survivin tosaid binding partner by selectively binding to the binding partner. 27.The method of claim 26 wherein said agent is a polypeptide fragment ofSurvivin.
 28. The method of claim 26 wherein said agent comprises thepolypeptide sequence EGWEPDDDPIEEHKKHSSGC, its conservativelysubstituted homologs and small molecule peptidomimetics thereof.
 29. Amethod for identifying agents that block the interaction of Survivinwith a Survivin binding partner comprising the steps of: a) incubatingSurvivin, or a fragment thereof, or a polypeptide comprising thesequence EGWEPDDDPIEEHKKHSSGC, and a binding partner, or a fragmentthereof, with an agent to be tested, and b) determining whether saidagent blocks the binding of Survivin to said Survivin binding partner.30. A method to assay for the presence of Survivin comprising the stepof determining whether a Survivin protein is expressed by a sample. 31.The method of claim 30, wherein said sample is selected from the groupconsisting of a tissue biopsy, stool, blood, urine and saliva.
 32. Themethod of claim 30 further comprising the steps of: a) preparing anextract of the cells in said sample, and b) examining the proteins ofsaid cell extract to determine the presence of a Survivin protein. 33.The method of claim 30 further comprising the steps of: a) preparing anextract of the cells in said sample, and b) examining the mRNA of saidcell extract to determine the presence of a Survivin encoding mRNA. 34.The method of claim 30 wherein said method is used to determine thegrowth potential of a tumor cell by correlating the level of Survivinexpression with control samples to indicate tumor growth potential. 35.A method of detecting or monitoring the stage or progression of a cancercomprising the steps of: obtaining a biological fluid sample from asubject; contacting said sample with a monoclonal antibody thatrecognizes and binds to Survivin; and determining whether the monoclonalantibody recognizes and binds to Survivin in said sample, the presenceof Survivin thereby predicting the presence of cancer.
 36. The method ofclaim 35, wherein the presence of Survivin predicts late stageneoplastic disease.
 37. A method for preserving the growth of cells inculture, comprising the step of contacting the cells with an amount ofSurvivin that is effective to reduce apoptosis.
 38. A kit for detectingthe presence of Survivin in a sample, comprising an antibody that bindsspecifically to Survivin and reagents to detect the antibody-Survivinbinding pair.
 39. A therapeutic vaccine that modulates the level ofSurvivin-mediated apoptosis in a host.
 40. The vaccine of claim 39,comprising a component that decreases the activity of Survivin in thehost, selected from the group of components consisting of: (1) Survivinprotein or fragments thereof sufficient to evoke a cellular mediatedresponse, (2) a DNA molecule that is antisense to Survivin mRNA orportions therof sufficient to inhibit translation of Survivin, (3) thesense DNA strand of EPR-1 acid or portions therof sufficient to inhibittranslation of Survivin.
 41. A method of prophylactic or preventativeanti-apoptotic therapy, comprising the administration of a Survivinpolypeptide, Survivin polypeptide fragment, an apoptosis-inhibitingpeptidomimetic thereof, a transgene encoding a Survivin polypeptide, ora transgene encoding a Survivin polypeptide fragment to a subject beforethe occurrence of a disease phenotype associated with cell apoptosis.42. The method of claim 41, wherein the disease phenotype is adiminished T cell count in a subject infected with HIV.
 43. The methodof claim 41, wherein the disease phenotype is a degenerative disease.44. The method of any of claim 6 and 20 to 22, further comprising theadministration of one or more additional agents to control cell growth.45. A method to treat cancer in a patient comprising the step ofincreasing the level of apoptosis in one or more cells of the cancer bydecreasing the amount or activity of Survivin in said one or more cancercells.
 46. The method of claim 45, further comprising the step ofadministering to the patient an agent that decreases the translation ofSurvivin mRNA in said one or more cancer cells, in an amount that iseffective to increase the level of apoptosis in said one or more cancercells.
 47. The method of claim 45, further comprising the step ofadministering to the patient an agent that decreases theapoptosis-inhibiting activity of the Survivin polypeptide present insaid one or more cancer cells, in an amount that is effective toincrease the level of apoptosis in the cell.
 48. The method of claim 47,wherein said decreased apoptosis-inhibiting activity of Survivin iseffected by the administration an agent that blocks the intracellularinteraction of Survivin with a Survivin binding partner.
 49. The methodof claim 48 wherein said agent blocks the binding of said Survivin tosaid binding partner by selectively binding to Survivin.
 50. The methodof claim 48 wherein said agent blocks the binding of said Survivin tosaid binding partner by selectively binding to said binding partner. 51.The method of claim 50 wherein said agent is a fragment of Survivin. 52.The method of claim 50 wherein said agent comprises the polypeptidesequence EGWEPDDDPIEEHKKHSSGC, its conservatively substituted homologsand small molecule peptidomimetics thereof.
 53. A method of inhibitingor reversing reperfusion injury in a subject comprising the step ofadministering to subject in need thereof a Survivin polypeptide, anapoptosis-inhibiting Survivin polypeptide fragment or anapoptosis-inhibiting peptidomimetic thereof.
 54. The method of claim 53wherein said step of administering comprises local administration at thesite of injury.
 55. A method for inhibiting or preventing tissue ororgan transplant rejection, comprising a local administration of aSurvivin polypeptide, an apoptosis-inhibiting Survivin polypeptidefragment, an apoptosis-inhibiting peptidomimetic thereof, a transgeneencoding a Survivin polypeptide or a transgene encoding anapoptosis-inhibiting Survivin polypeptide fragment to the tissue ororgan or to a site proximal to the transplant.
 56. A method forenhancing viability of organs and tissues prior to their transplantationinto a subject, comprising the perfusion of the organs or tissues with aa Survivin polypeptide, an apoptosis-inhibiting Survivin polypeptidefragment, or an apoptosis-inhibiting peptidomimetic thereof.
 57. Themethod of claim 56, wherein said transgene is delivered via a viralvector.
 58. The method of claim 57 wherein said vector is replicationdefective.
 59. The method of claim 56 wherein said transgene isdelivered as a naked nucleic acid.